Radioligands for myelin

ABSTRACT

A radioligand for labeling myelin includes a fluorescent trans-stilbene derivative.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application Nos.62/305,769, filed Mar. 9, 2016 and 62/445,555 filed Jan. 12, 2017, thesubject matter of which are incorporated herein by reference in theirentirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. NS061837awarded by the National Institute of Health (NIH). The United Statesgovernment has certain rights to the invention.

TECHNICAL FIELD

Embodiments described herein relate to radioligands and to methods oftheir use, and particularly relates to radioligands that readily enterthe brain and selectively localize in the myelinated regions.

BACKGROUND

Myelin is a specialized membrane that ensheathes neuronal axons,promoting efficient nerve impulse transmission (Morell and Quarles(1999) Basic Neurochemistry: molecular, cellular, and medical aspects.In Siegel G J, ed. Myelin Formation, Structure, and Biochemistry.Lippincott-Raven Publishers, 79-83). Due to its important biologicalfunctions in the normal central nervous system (CNS) and itsvulnerability in disease, several techniques have been developed tovisualize and characterize myelin histopathology. These can be broadlydivided into those based upon antibody immunohistochemistry (IHC)(Horton and Hocking (1997) Cereb. Cortex 7:166-177) and more traditionalhistochemical procedures. The classic histochemical stains include luxolfast blue MBN (Kluver and Barrera (1953) J Neurosci Methods 153:135-146; Presnell and Schreibman (1997) Humanson's Animal TissueTechniques, 5^(th) ed.; Kiernan (1999) Histological and HistochemicalMethods: Theory and practice, 3^(rd) ed.; Bancroft and Gamble (2002),Theory and Practice of Histological Techniques, 5 ed. and Sudan Black B(Lison and Dagnelie (1935) Bull. d'Histologie Appliquee 12: 85-91).Traditional chromogenic methods also include the Palweigert method((Weigert (1884) Fortschr Deutsch Med 2: 190-192, (1885) FortschrDeutsch Med 3:236-239; Clark and Ward (1934) Stain Technol 54:13-16),the Weil stain (Weil (1928) Arch Neurol Psychiatry 20:392-393; Berube etal. (1965) Stain Technol 40:53-62)), the Loyez method (Cook (1974)Manual of Histological Demonstration Methods, 5^(th) ed.), and a methodbased on horse serum followed by subsequent reaction withdiaminobenzidine (McNally and Peters (1998) J Histochem Cytochem46:541-545). In addition, modified silver stains including the Gallyasmethod (Pistorio et al. (2005) J Neurosci Methods 153: 135-146) andSchmued's gold chloride technique (Schmued and Slikker (1999) Brain Res837:289-297) have also been used as simple, high-resolutionhistochemical markers of myelin. More recently, fluoromyelin (Kanaan etal. (2005) Am J Physiol Regul Integr Comp Physiol 290:R1105-1114) andNIM (Xiang et al. (2005) J Histochem Cytochem 53:1511-1516) wereintroduced as novel myelin dyes, which enable quick and selectivelabeling of myelin in brain tissue sections. Although thesemyelin-staining techniques are widely used in vitro, none can be appliedin vivo due to impermeability of the blood-brain barrier (BBB). The lackof in vivo radioligands has limited the progress of myelin imaging andhindered efficacy evaluation of novel myelin repair therapies duringtheir development.

SUMMARY

Embodiments described herein relate to radioligands for use in thedetection and quantification of myelin in a subject. The radioligandsinclude a fluorescent trans-stilbene derivative having the followingformula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup;

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

R³ and R⁴ are same or different and are each independently H, NHR″,where R″ is H or a lower alkyl group, or a radiolabeled lower alkylgroup, alkylene group, alkenyl group, alkynyl group, or alkoxy group;

R⁵ and R⁶ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

R⁷ and R⁸ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

wherein at least one of R¹, R², R³, or R⁴ includes a radiolabel selectedfrom the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or apharmaceutically acceptable salt thereof. For example, the at least oneradiolabel can be ¹⁸F.

Other embodiments described herein relate to a method of detectingmyelin in a subject's tissue. The myelin can be associated with nervesof the central system and/or the peripheral system. The tissue caninclude brain tissue, spinal tissue, and other tissue associated withthe peripheral nervous system. In some aspects, the tissue can bemyelinated tissue at a surgical site.

The method includes administering to the subject a radioligand thatincludes a fluorescent trans-stilbene derivative having the followingformula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup;

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

R³ and R⁴ are same or different and are each independently H, NHR″,where R″ is H or a lower alkyl group, or a radiolabeled lower alkylgroup, alkylene group, alkenyl group, alkynyl group, or alkoxy group;

R⁵ and R⁶ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

R⁷ and R⁸ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

wherein at least one of R¹, R², R³, or R⁴ includes a radiolabel selectedfrom the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or apharmaceutically acceptable salt thereof.

Following, administration of the radioligand, the radioligand can bedetected to determine the location, distribution, and/or amount of theradioligand that is bound to and/or labels the myelin.

Other embodiments described herein relate to a method of detecting amyelination related disorder in a subject. The method includesadministering to the subject a radioligand that includes a fluorescenttrans-stilbene derivative having the following formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup;

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

R³ and R⁴ are same or different and are each independently H, NHR″,where R″ is H or a lower alkyl group, or a radiolabeled lower alkylgroup, alkylene group, alkenyl group, alkynyl group, or alkoxy group;

R⁵ and R⁶ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

R⁷ and R⁸ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

wherein at least one of R¹, R², R³, or R⁴ includes a radiolabel selectedfrom the group consisting of ¹⁸F ¹²³I, ¹²⁵I, and ^(99m)Tc; or apharmaceutically acceptable salt thereof.

The distribution and/or amount of the radioligand bound to and/orlabeling myelin in the subject's neural tissue is then detected,measured, and/or quantified and compared to a control. A decreaseddistribution and/or amount of the radioligand compared to the controlcan be indicative of a decrease in myelination of the neural tissue.

In some aspects, the myelination related disorder includes aneurodegenerative autoimmune disease. In certain aspects, theneurodegenerative disease can be multiple sclerosis.

Still other embodiments relate to a method of monitoring the efficacy ofa remyelination therapy in a subject. The method includes administeringto a subject undergoing remyelination therapy a radioligand thatincludes a fluorescent trans-stilbene derivative having the followingformula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup;

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

R³ and R⁴ are same or different and are each independently H, NHR″,where R″ is H or a lower alkyl group, or a radiolabeled lower alkylgroup, alkylene group, alkenyl group, alkynyl group, or alkoxy group;

R⁵ and R⁶ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

R⁷ and R⁸ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

wherein at least one of R², R³, or R⁴ includes a radiolabel selectedfrom the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or apharmaceutically acceptable salt thereof.

The distribution and/or amount of the radioligand bound to and/orlabeling myelin in the subject's neural tissue is detected, measured,and/or quantified and compared to a control. An increased distributionand/or amount of the radioligand compared to the control can beindicative of efficacy of the remyelination therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill become apparent to those skilled in the art to which the presentinvention relates upon reading the following description with referenceto the accompanying drawings.

FIG. 1 illustrates a plot showing excitation (Em 420±10 nm; solid) andemission (Ex 390±10 nm; dashed) spectra of the compounds 25, 32-37 and39.

FIGS. 2(A-B) illustrate an image and graph showing FIR in white mattervs gray matter. (A) Representative in vitro tissue staining showing ROIused for calculation for FIR between white matter and gray matter. (B)ImageJ was used to calculate FIR of each target compound showingcompounds 32 and 35 with FIR >2.

FIGS. 3(A-D) illustrate ex vivo images of corpus callosum and the wholebrain with compounds (A and B) 32 and (C and D) 35.

FIGS. 4(A-B) illustrate plots and a graph showing (A) Averageradioactivity concentration of target compounds in the whole brain interms of SUV as a function of time. (B) Average SUV of late time points(40-60 min).

FIGS. 5(A-C) illustrate images showing representative (A) coronal, (B)sagittal, and (C) axial microPET/CT fusion images of the rat brainfollowing i.v. administration of [¹⁸F]32, showing high uptakes of[18F]32 in the white matter region of the brain.

FIGS. 6(A-E) illustrate film autoradiography. (A) Ex vivoautoradiography showing [¹⁸F]32 binds to myelinated corpus callosum (CC)in mouse brain (coronal) and was consistent with histological stainingof myelinated regions (C). (B) After pretreatment with nonlabeled CIC,ex vivo autoradiographic visualization of CC was significantlydecreased. Distinct staining of CIC was observed when the same sectionwas viewed under fluorescent microscope (D). (E) Statistical analysis ofoptical density ratio of gcc to cortex on the film showed there issignificant difference between control and ex vivo or in vitro blockstudies. *: p<0.05.

FIG. 7 illustrates plots showing excitation (Ex) and emission (Em)spectra of compounds 18-23 (0.3 mM in methylene chloride). Excitationspectra scans from 300 to 415 nm and emission spectra scans from 394 to600 nm. Bandwidth at 5 nm, scan at 120 nm/min, and integration time of0.5 s. Maximal excitation wavelengths of compounds 18-23 were at 375 and395 nm, while maximal emission wavelengths of compounds 18-23 were at430 nm.

FIGS. 8(A-G) illustrate images showing tissue staining of compound 21 inwild-type mouse brain and demyelination LPC rat model brain, andpreliminary compound screening with FIR calculated based on ROIs. Freshfrozen wild-type mouse brain sections were stained with compound 21, andhighly myelinated corpus callosum (A) and cerebellum myelin track (B)were clearly visualized. In vitro tissue staining showed that compound21 can be used to detect demyelinated lesion present in a rat brain ofLPC model (C), which is further confirmed by LFB and cresyl violetstaining (D) on an adjacent tissue section. In situ staining proved thatcompound 21 crosses the BBB and stains myelinated areas of the mousebrain (E) and spinal cord (F). Representative in vitro tissue stainingof coronal sections showing ROIs used for calculation of FIRs betweenwhite matter and gray matter (G), and FIRs of compounds 18-23 werecalculated by ImageJ (H).

FIGS. 9(A-E) illustrate images and plots showing representative (A)coronal, (B) sagittal, and (C) axial microPET/CT fusion images of therat brain following iv administration of [¹⁸F]21, showing high uptake inthe white matter region of the brain. (D) Quantitative analysis ofaverage radioactivity concentration of target compound [¹⁸F]21 in thewhole brain of rats (n=3) in terms of SUV as a function of time. (E) Insitu autoradiography showing [¹⁸F]21 binds to myelinated corpus callosumin mouse brain (coronal).

FIGS. 10(A-B) illustrates representative coronal microPET images of theshiverer mouse brain (A) and WT mouse brain (B) following ivadministration of [¹⁸F]21, showing higher brain uptake in the controlthan that in the shiverer mouse. Quantitative analysis of total uptakeof [¹⁸F]21 in terms of SUV at 40-60 min post-injection in shiverer mice[Shi] and control mouse [WT], showing significant higher uptake in WTmouse brain (p=0.00028, two-tailed t test).

FIGS. 11(A-E) illustrate MicroPET/CT images. Representative PET/CTfusion images in rats acquired before SCI surgery (baseline scan) withhigher magnification coronal (A) and sagittal images (B) in the T13spinal cord. Representative PET/CT fusion images in rats acquired 1 dayafter SCI surgery with higher magnification coronal (C) and sagittalimages (D) in the T13 spinal cord. Note the decrease signal contrast inthe SCI animal compared with the baseline scan. (E) Quantification ofthe total cumulative [¹⁸F]21 uptake in the T13 ROI 30-60 post injectionshowed significantly lower uptake in the SCI group compared withbaseline scans.

FIGS. 12(A-B) illustrate in situ histological staining of the SCI (T13)tissue section with reference compound 21 after microPET/CT imagingshowing a demyelinated lesion at dorsal portion (A), which is consistentwith LFB and cresyl violet staining using adjacent sections (B).

FIGS. 13(A-D) illustrate coregistration of in situ 3D fluorescenceimaging with microPET/CT imaging. (A) [¹⁸F]21 PET/CT imaging of spinalcord in SCI rat showing reduced uptake after contusive injury at T13 bythe arrowhead. (B) 3D in situ fluorescence images of the spinal cordcoregistered with CT image of the spinal cord in SCI rat showing reducedfluorescence intensity at T13 by the arrowhead. (C) Fusion images ofPET, CT, and 3D fluorescence images. (D) Prior to 3D coregistration, asagittal slice of the 3D fluorescence image stack indicatesdemyelination corresponding with microPET/CT imaging.

DETAILED DESCRIPTION

The definitions provided herein are to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the application.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. When the term “pharmaceutically acceptable” isused to refer to a pharmaceutical carrier or excipient, it is impliedthat the carrier or excipient has met the required standards oftoxicological and manufacturing testing or that it is included on theInactive Ingredient Guide prepared by the U.S. Food and Drugadministration. “Pharmacologically active” (or simply “active”) as in a“pharmacologically active” derivative or analog, refers to a derivativeor analog having the same type of pharmacological activity as the parentcompound and approximately equivalent in degree.

The term “pharmaceutically acceptable salts” or complexes refers tosalts or complexes that retain the desired biological activity of theparent compound and exhibit minimal, if any, undesired toxicologicaleffects. Non-limiting examples of such salts are (a) acid addition saltsformed with inorganic acids (for example, hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid, and the like), andsalts formed with organic acids such as acetic acid, oxalic acid,tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid,tannic acid, palmoic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acids, naphthalenedisulfonic acids, andpolygalacturonic acid; (b) base addition salts formed with cations suchas sodium, potassium, zinc, calcium, bismuth, barium, magnesium,aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and thelike, or with an organic cation formed fromN,N-dibenzylethylene-diamine, ammonium, or ethylenediamine; or (c)combinations of (a) and (b); e.g., a zinc tannate salt or the like.

A radioligand exhibits “specific binding” or “selective binding” tomyelin if it associates more frequently with, more rapidly with, for alonger duration with, or with greater affinity to, myelin than withtissues not containing myelin. “Non-specific binding” refers to bindingof the radioligand to non-myelin containing tissue. For relative bindingvalues, such as specific binding or nonspecific binding, each sampleshould be measured under similar physical conditions (i.e., temperature,pH, and solvent). Generally, specific binding is characterized by arelatively high affinity of a radioligand to a receptor and a relativelylow to moderate capacity. Typically, binding is considered specific whenthe affinity constant Ka is at least 10⁶ M⁻¹. A higher affinity constantindicates greater affinity, and thus typically greater specificity.“Non-specific” binding usually has a low affinity with a moderate tohigh capacity. Non-specific binding usually occurs when the affinityconstant is below 10⁶M⁻¹.

The phrase “parenteral administration” refers to any means ofintroducing a substance or compound into a subject, that does notinvolve oral ingestion or direct introduction to the gastrointestinaltract, including but not limited to subcutaneous injection,intraperitoneal injection, intramuscular injection, intravenousinjection, intrathecal injection, intracerebral injection,intracerebroventricular injection, or intraspinal injection, or anycombination thereof.

The phrase “remyelination” refers to the spontaneous, therapeutic, orexperimentally induced repair, regeneration, or otherwise enhancedconstitution or functionality of the insulating material ensheathingneuronal axons.

The phrase “molecular imaging” refers to a non-invasive technique for invivo imaging of biological targets at molecular level. Molecular imagingcan involve the targeting of a biomarker with a radioligand.

The phrase “radioligand” refers to a compound that specifically binds toa biomarker (e.g., myelin), allowing for the imaging and studying of themarker. As used herein, the phrase “biomarker” refers to a biologicalsubstance that is specific to a certain biological process or mechanism.

The term “subject” refers to an animal, such as a mammal including asmall mammal (e.g., mouse, rat, rabbit, or cat) or a larger mammal(e.g., dog, pig, or human). In particular aspects, the subject is alarge mammal, such as a human.

The terms “control” or “control sample” refer to one or more biologicalsamples isolated from an individual or group of individuals that arenormal (i.e., healthy). The term “control”, “control value” or “controlsample” can also refer to the compilation of data derived from samplesof one or more individuals classified as normal, one or more individualsdiagnosed with a myelination related disease.

The phrase “having the formula” or “having the structure” is notintended to be limiting and is used in the same way that the term“comprising” is commonly used.

The term “alkyl” refers to a branched or unbranched saturatedhydrocarbon group typically although not necessarily containing 1 toabout 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well ascycloalkyl groups, such as cyclopentyl, cyclohexyl, and the like.Generally, although again not necessarily, alkyl groups herein contain 1to about 18 carbon atoms, preferably 1 to about 12 carbon atoms. Theterm “lower alkyl” intends an alkyl group of 1 to 6 carbon atoms.Substituents identified as “C₁-C₆ alkyl” or “lower alkyl” can contain 1to 3 carbon atoms, and more particularly such substituents can contain 1or 2 carbon atoms (i.e., methyl and ethyl). “Substituted alkyl” refersto alkyl substituted with one or more substituent groups, and the terms“heteroatom-containing alkyl” and “heteroalkyl” refer to alkyl in whichat least one carbon atom is replaced with a heteroatom, as described infurther detail infra. If not otherwise indicated, the terms “alkyl” and“lower alkyl” include linear, branched, cyclic, unsubstituted,substituted, and/or heteroatom-containing alkyl or lower alkyl,respectively.

The term “alkenyl” refers to a linear, branched or cyclic hydrocarbongroup of 2 to about 24 carbon atoms containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl,and the like. Generally, although again not necessarily, alkenyl groupscan contain 2 to about 18 carbon atoms, and more particularly 2 to 12carbon atoms. The term “lower alkenyl” refers to an alkenyl group of 2to 6 carbon atoms, and the specific term “cycloalkenyl” intends a cyclicalkenyl group, preferably having 5 to 8 carbon atoms. The term“substituted alkenyl” refers to alkenyl substituted with one or moresubstituent groups, and the terms “heteroatom-containing alkenyl” and“heteroalkenyl” refer to alkenyl in which at least one carbon atom isreplaced with a heteroatom. If not otherwise indicated, the terms“alkenyl” and “lower alkenyl” include linear, branched, cyclic,unsubstituted, substituted, and/or heteroatom-containing alkenyl andlower alkenyl, respectively.

The term “alkynyl” refers to a linear or branched hydrocarbon group of 2to 24 carbon atoms containing at least one triple bond, such as ethynyl,n-propynyl, and the like. Generally, although again not necessarily,alkynyl groups can contain 2 to about 18 carbon atoms, and moreparticularly can contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6 carbon atoms. The term “substitutedalkynyl” refers to alkynyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkynyl” and“heteroalkynyl” refer to alkynyl in which at least one carbon atom isreplaced with a heteroatom. If not otherwise indicated, the terms“alkynyl” and “lower alkynyl” include linear, branched, unsubstituted,substituted, and/or heteroatom-containing alkynyl and lower alkynyl,respectively.

The term “alkoxy” refers to an alkyl group bound through a single,terminal ether linkage; that is, an “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. A “lower alkoxy” group intendsan alkoxy group containing 1 to 6 carbon atoms, and includes, forexample, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc.Preferred substituents identified as “C₁-C₆ alkoxy” or “lower alkoxy”herein contain 1 to 3 carbon atoms, and particularly preferred suchsubstituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

The term “cyclic” refers to alicyclic or aromatic substituents that mayor may not be substituted and/or heteroatom containing, and that may bemonocyclic, bicyclic, or polycyclic.

By “substituted” as in “substituted alkyl,” “substituted aryl,” and thelike, as alluded to in some of the aforementioned definitions, is meantthat in the alkyl, aryl, or other moiety, at least one hydrogen atombound to a carbon (or other) atom is replaced with one or morenon-hydrogen substituents. In addition, the aforementioned functionalgroups may, if a particular group permits, be further substituted withone or more additional functional groups or with one or more hydrocarbylmoieties such as those specifically enumerated above. Analogously, theabove-mentioned hydrocarbyl moieties may be further substituted with oneor more functional groups or additional hydrocarbyl moieties such asthose specifically enumerated.

When the term “substituted” appears prior to a list of possiblesubstituted groups, it is intended that the term apply to every memberof that group. For example, the phrase “substituted alkyl, alkenyl, andaryl” is to be interpreted as “substituted alkyl, substituted alkenyl,and substituted aryl.” Analogously, when the term“heteroatom-containing” appears prior to a list of possibleheteroatom-containing groups, it is intended that the term apply toevery member of that group. For example, the phrase“heteroatom-containing alkyl, alkenyl, and aryl” is to be interpreted as“heteroatom-containing alkyl, substituted alkenyl, and substitutedaryl.”

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present on a given atom, and,thus, the description includes structures wherein a non-hydrogensubstituent is present and structures wherein a non-hydrogen substituentis not present.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

This application relates to radioligands that upon administration to asubject or tissue of the subject can specifically or selectively bindto, localize with, and/or label myelinated regions of the nervoussystem, including the central nervous system and peripheral nervoussystem, and upon specifically or selectively binding to, localizingwith, and/or labeling can be imaged, using, for example, fluorescenceand/or positron emission tomography (PET) imaging, to detect, measure,and/or quantify the amount, level, and/or distribution of myelin in thetissue being imaged. The radioligands can bind to myelin membrane and donot bind to a component of degenerating myelin fragments. Theradioligands can also be readily visualized using conventionalvisualization techniques to indicate myelinated regions of the brain,central nervous system, and peripheral nervous system. The radioligandsdescribed herein can be used in a method of detecting or quantifying alevel of myelination in vivo in a subject, a method of detecting amyelination related disorder in a subject, a method of monitoring theremyelination effects of an agent in a subject, a method of screeningthe myelination effects of an agent in a subject, surgical methods wherethe presence or location of nerves is desired, and multi-modal imagingapplications of myelin.

The radioligand can be detected in vivo using positron emissiontomography (PET) as well as by fluorescent imaging. PET is a functionalimaging technique that can detect chemical and metabolic change at themolecular level. Another example of an in vivo imaging modality that canbe used to detect a radioligand is MicroPET. MicroPET is a highresolution positron emission tomography scanner designed for imagingsmall laboratory animals. In some aspects of the invention, the in vivoimaging modality is single-photon emission computerized tomography(SPECT).

In an embodiment of the application, the radioligands can exhibitexcitation wavelengths in a range of about 370 nm to about 400 nm andemission wavelengths in a range of about 410 nm to about 440 nm. Thus,the radioligands can be used in a method for irradiating and imagingmyelin with light of the wavelength range from about 370 nm to about 440nm. For example, a radioligand described herein can have excitationpeaked at about 390 nm and emission peaked at about 420 nm.

In some aspects, the radioligands can meet the requirements thatgenerally apply to diagnostic pharmaceuticals. As these substances maybe applied at higher doses and for a longer diagnostic period, they canhave a low-toxicity. In addition, the radioligands described herein canbe of low molecular weight, lipophilic, and readily penetrate theblood-brain barrier in sufficient amounts to be detectable by positronemission tomography imaging and bind to myelin fibers with high affinityand specificity without being rapidly degraded. In some embodiments, theradioligands can have a lipophilicity of about 2.5 to about 5.4 or about2.7 to about 4.0 to enhance crossing of the blood brain barrier uponsystemic administration of the radioligand to the subject. Theradioligands are also sufficiently stable in chemical and photophysicalrespect, at least for as long as the diagnostic period lasts.

The radioligand can include a fluorescent trans-stilbene derivative or apharmacophore thereof (e.g., coumarin pharmacophore) that is less thanabout 700 daltons and has a relatively high binding affinity (Kd) (e.g.,at least about 1.0 nM) to isolated myelin fractions but a relatively lowbinding affinity (Kd) to isolated non-myelin fractions. The terms“fluorescent trans-stilbene” or “fluorescent trans-stilbene derivative”or “fluorescent trans-stilbene compound” are meant encompass not onlythe specified molecular entity but also its pharmaceutically acceptable,pharmacologically active analogs, including, but not limited to, salts,esters, amides, prodrugs, conjugates, active metabolites, and other suchderivatives, analogs, and related compounds.

In some embodiments, the radioligand can include a fluorescenttrans-stilbene derivative having the following formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup;

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

R³ and R⁴ are same or different and are each independently H, NHR″,where R″ is H or a lower alkyl group, or a radiolabeled lower alkylgroup, alkylene group, alkenyl group, alkynyl group, or alkoxy group;

R⁵ and R⁶ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

R⁷ and R⁸ are H or are linked to form a cyclic ring, wherein the cyclicring is aromatic, alicyclic, heteroaromatic, or heteroalicyclic;

wherein at least one of R², R³, or R⁴ includes a radiolabel selectedfrom the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or apharmaceutically acceptable salt thereof. For example, the at least oneradiolabel can be ¹⁸F.

In other embodiments, the radioligand can include a fluorescent stilbenederivative having the following formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup;

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

R³ is H, or a radiolabeled lower alkyl group, alkylene group, alkenylgroup, alkynyl group, or alkoxy group;

wherein at least one of R′, R², or R³ includes a radiolabel selectedfrom the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or apharmaceutically acceptable salt thereof. For example, the at least oneradiolabel can be ¹⁸F.

In other embodiments, the radioligand can include a fluorescent stilbenederivative having the following formula:

wherein R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group;

R³ is H, or a radiolabeled lower alkyl group, alkylene group, alkenylgroup, alkynyl group, or alkoxy group;

wherein at least one of R² or R³ includes a radiolabel selected from thegroup consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or a pharmaceuticallyacceptable salt thereof. For example, the at least one radiolabel can be¹⁸F.

In other embodiments, the radioligand can include a fluorescent stilbenederivative having the following formula:

wherein R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group;

X¹ is a lower alkyl group, alkylene group, alkenyl group, alkynyl group,or alkoxy group;

Y¹ is a radiolabel selected from the group consisting of ¹⁸F, ¹²³I,¹²⁵I, and ^(99m)Tc; or a pharmaceutically acceptable salt thereof. Forexample, the radiolabel can be ¹⁸F.

In other embodiments, the radioligand can include a fluorescent stilbenederivative having the following formula:

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

n¹ is 1 to 6;

Y¹ is a radiolabel selected from the group consisting of ¹⁸F, ¹²³I,¹²⁵I, and ^(99m)Tc; or a pharmaceutically acceptable salt thereof. Forexample, the radiolabel can be ¹⁸F.

In other embodiments, the radioligand can include a fluorescent stilbenederivative having the following formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup;

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

wherein at least one of R¹ or R² includes a radiolabel selected from thegroup consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or a pharmaceuticallyacceptable salt thereof. For example, the radiolabel can be ¹⁸F.

In other embodiments, the radioligand can include a fluorescent stilbenederivative having the following formula:

wherein R² is H, or NHR′, where R′ is H or a lower alkyl group;

n² is 1 to 6;

n³ is 1 to 6;

Y¹ is a radiolabel selected from the group consisting of ¹⁸F, ¹²³I,¹²⁵I, and ^(99m)Tc; or a pharmaceutically acceptable salt thereof. Forexample, the radiolabel can be ¹⁸F.

In other embodiments, the radioligand can include a fluorescent coumarinderivative that is a pharmacophore of trans-stilbene. The fluorescentcoumarin derivative can have the following formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup;

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

R³ and R⁴ are same or different and are each independently H, NHR″,where R″ is H or a lower alkyl group, or a radiolabeled lower alkylgroup, alkylene group, alkenyl group, alkynyl group, or alkoxy group;

wherein at least one of R¹, R², R³, or R⁴ includes a radiolabel selectedfrom the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or apharmaceutically acceptable salt thereof. For example, the radiolabelcan be ¹⁸F.

In other embodiments the radioligand is fluorescent coumarin derivativehaving the following formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup;

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group;

wherein at least one of R¹ or R² includes a radiolabel selected from thegroup consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or a pharmaceuticallyacceptable salt thereof. For example, the radiolabel can be ¹⁸F.

In other embodiments the radioligand can include a fluorescent coumarinderivative having the following formula:

wherein R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group;

R³ and R⁴ are same or different and are each independently H, NHR″,where R″ is H or a lower alkyl group, or a radiolabeled lower alkylgroup, alkylene group, alkenyl group, alkynyl group, or alkoxy group;

wherein at least one of R², R³, or R⁴ includes a radiolabel selectedfrom the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or apharmaceutically acceptable salt thereof. For example, the radiolabelcan be ¹⁸F.

In further embodiments, the radioligand can be selected from thefollowing structures:

or pharmaceutically acceptable salts thereof.

The foregoing formulae represent the general structures of radiolabeledfluorescent trans-stilbene compounds found to be effective radioligandsfor labeling myelin in vivo as well as in vitro as described in theexamples below. They are characterized by their ability to beadministered to a mammal or subject parenterally and selectivelylocalize to myelinated regions in the brain, central nervous system, andperipheral nervous system via direct binding to myelin membranes and notbind to degenerating myelin fragments.

By way of example, the radioligands can be administered to white matterand grey matter samples of mouse brain and the fluorescent intensity ofthe samples can be measured by fluorescent microscopy staining. Thefluorescent intensity ratio (FIR) in the same region of interest (ROI)between white matter and grey matter can be calculated. The FIR of thefluorescent trans-stilbene derivatives described herein can be at leastabout 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0.

The radioligands described herein are unique in that they exhibitnegligible toxicities as demonstrated in both preclinical and clinicalsettings, making them suitable candidates for clinical imagingmodalities and translational studies. For example, the radioligands canbe used for positron emission tomography to detect and quantify myelincontents in vivo.

Typically, the radioligand can be formulated into a pharmaceuticalcomposition prior to use. When a composition described herein is appliedto a subject, it is formulated to be compatible with its intended routeof application. Examples of routes of application include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, DMSO, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid orcyclodextrin; buffers such as acetates, citrates or phosphates andagents for the adjustment of tonicity such as sodium chloride ordextrose. pH can be adjusted with acids or bases, such as hydrochloricacid or sodium hydroxide.

In one example, a radioligand solution includes a 10 mM radioligandsolution. A radioligand solution can also contain saline, DMSO, and HCL.One skilled in the art can utilize the radioligand with pharmaceuticalcarriers and/or excipients in varying concentrations and formulationsdepending on the desired use.

In certain embodiments, the radioligands described herein can becontacted with or administered to a subject's brain tissue, centralnervous system, and/or peripheral nervous system and utilized forlabeling and detecting myelinated regions of the subject's brain tissue,central nervous system, and/or peripheral nervous system. Theradioligand may be administered to the subject's nervous or neuraltissue either in vivo or in vitro after a tissue sample has been removedfrom the body. Myelinated regions of the subject's brain are typicallyfound in the white matter of the brain in the myelin sheaths of neuronalaxons. Myelin is an outgrowth of glial cells, more specificallyoligodendrocytes, which serve as an electrically insulating phospholipidlayer surrounding axons of many neurons. For purposes of the presentinvention, a subject's brain tissue is typically a mammal's braintissue, such as a primate, e.g., chimpanzee or human; cow; dog; cat; arodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; orfish.

In some embodiments, the radioligands described herein can be used forthe in vivo detection and localization of myelinated regions of ananimal's brain, central nervous system, and/or peripheral nervoussystem. The radioligand can be administered to the subject or animal asper the examples contained herein, but typically through intravenousinjection. “Administered”, as used herein, means provision or deliveryradioligands in an amount(s) and for a period of time(s) effective tolabel myelin in an animal's brain, central nervous system, and/orperipheral nervous system. The administration of a compound orcomposition described herein to a subject may be systemically orlocally. For example, a radioligand described herein may be administeredsystemically, (e.g., parenterally or intravenously) to the subject suchthat it is delivered throughout the body. Parenteral route includesintravenous, intramuscular, intraperitoneal, intrasternal, andsubcutaneous injection or infusion.

An example of a dosing regimen is to administer about 40 to about 50mg/kg by weight to the animal. In one example at 5 min, the brainconcentration of radioligand can range between about 4% to 24% ID/g toensure sufficient detection of the myelinated regions of the brain,central nervous system, and/or peripheral nervous system.

In some embodiments, the radioligands can be used in analytical,diagnostic, or prognostic applications related to myelin detection. Forexample, researchers studying normal brains can employ radioligands andmethods described herein to examine the morphology and distribution ofmyelinated tissue in a subject. The radioligands and methods are alsoapplicable in intraoperative nerve labeling, spinal imaging,non-invasive in vivo measurement of myelination levels, and preclinicaland basic neuroscience bench research aimed at the study of the functionand process of myelination, and the dysfunction and repair of myelin. Insome embodiments, researchers studying normal nervous system tissue canemploy this method to examine the morphology and distribution ofmyelinated tissue in an animal.

“Distribution” as used herein is the spatial property of being scatteredabout over an area or volume. In this case the “distribution ofmyelinated tissue” is the spatial property of myelin being scatteredabout over an area or volume included in the animal's brain, centralnervous system, or peripheral nervous system tissue. Researchersinterested in neurotoxicology and neuropathology can also use thismethod in several ways. One way is to infer demyelination by the absenceof the radioligand labeling compared to normal control tissue (e.g.,normal brain). A second way is to study morphological changes in themyelin such as a fragmented or beaded appearance of the myelin sheath.

In yet another embodiment, one skilled in the art can assess andquantify changes in myelin content in vivo. Myelin in a subject's tissue(e.g., brain) can be visualized and quantified using fluorescence or PETimaging. For quantitative analysis, the images are analyzed on a regionof interest basis. The radioligand may be visualized any time postadministration depending on the application. In one example, at 2 minpost administration, signals of the radioligand in proportion to themyelin content in a subject's brain is recorded. In another example,signals are recorded at about 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50,60, 70, 80, 90, 100, 110 and/or 120 min post-administration.

In other embodiments, myelin in an animal's brain, central nervoussystem, and/or peripheral nervous system can be visualized andquantified using an fluorescence or PET imaging modality. Theradioligand may be visualized any time post administration depending onthe application as typical radioligands embodied described herein have alow clearance rate due to specific binding in the myelinated regions(e.g., at 60 min, the brain concentration of probe can be ≤50% of 5 minvalue to ensure that half time retention in normally myelinated brain is60 min or longer).

For directly monitoring myelin changes in the white matter of a subject,radioligands described herein can readily penetrate the blood-brainbarrier (BBB) and directly bind to the myelinated white matter inproportion to the extent of myelination. Radioligands described hereincan be used in conjunction with PET as imaging markers to directlyassess the extent of total lesion volumes associated with demyelination.This can provide a direct clinical efficacy endpoint measure of myelinchanges and identify effective therapies aimed at protection and repairof axonal damages.

In some embodiments, the radioligand can include an additional imagingmoiety that allows the radioligand to be detected by other imagingmodalities, such as magnetic resonance imaging. This allows theradioligand to be used in a multi-modal imaging system and provide moresensitive and specific detection of myelin or myelination in tissue of asubject.

The additional imaging moiety can include a magnetic resonance contrastagent that is conjugated, coupled, or bound to an atom of theradioligand and facilitates detection of the radioligand by magneticresonance imaging. In some aspects, the magnetic resonance contrastagent can include a chelating group, such as a Gd chelating ligand toimprove the MRI contrast properties of the radioligand. In one example,as disclosed in U.S. Pat. No. 7,351,401, which is herein incorporated byreference in its entirety, the chelating group can be of the form W-L orV-W-L, wherein V is selected from the group consisting of —COO—, —CO—,—CH₂O— and —CH₂NH—; W is —(CH₂)n where n=0, 1, 2, 3, 4, or 5; and L is:

wherein M is selected from the group consisting of Tc and Re; or

wherein each R₃ is independently is selected from one of:

or a myelin binding, chelating compound (with or without a chelatedmetal group) or a water soluble, non-toxic salt thereof of the form:

wherein each R₃ independently is selected from one of:

The chelating group can be coupled to at least one atom of theradioligand through a carbon chain link. The carbon chain link cancomprise, for example about 2 to about 10 methylene groups and have aformula of, for example, (CH₂)_(n), wherein n=2 to 10.

The radioligands can also be used to diagnose a myelination relateddisorder in an animal through the use of in vivo myelin labeling. Thus,in certain embodiments, solutions containing the radioligands describeherein can be used in the detection of myelin related disorders in ananimal.

Methods of detecting a myelin related disorder include the steps oflabeling myelin in vivo in the animal's brain tissue with a radioliganddescribed herein, visualizing a distribution of the radioligand in theanimal's brain tissue as described above and in the examples, and thencorrelating the distribution of the radioligand with a myelin relateddisorder in the animal. In one example of detecting a myelin relateddisorder, the methods described herein can be used to compare myelinatedaxonal regions of the brain in the normal tissues of control populationsto those of a suspect animal. If the suspect animal has a myelin relateddisorder, myelin may be virtually absent in lesioned areas thusindicating the presence of a myelin related disorder.

Myelination disorders can include any disease, condition (e.g., thoseoccurring from traumatic spinal cord injury and cerebral infarction), ordisorder related to demyelination, remyelination, or dysmyelination in asubject. A myelin related disorder as used herein can arise from amyelination related disorder or demyelination resulting from a varietyof neurotoxic insults. Demyelination is the act of demyelinating, or theloss of the myelin sheath insulating the nerves, and is the hallmark ofsome neurodegenerative autoimmune diseases, including multiplesclerosis, transverse myelitis, chronic inflammatory demyelinatingpolyneuropathy, and Guillain-Barre Syndrome. Leukodystrophies are causedby inherited enzyme deficiencies, which cause abnormal formation,destruction, and/or abnormal turnover of myelin sheaths within the CNSwhite matter. Both acquired and inherited myelin disorders share a poorprognosis leading to major disability. Thus, some embodiments of thepresent invention can include methods for the detection ofneurodegenerative autoimmune diseases in an animal and more specificallythe detection of multiple sclerosis in an animal.

Another embodiment relates to a method of monitoring the efficacy of aremyelination therapy in an animal. Remyelination is the repair ofdamaged or replacement of absent myelin in an animal's brain tissue. Themethods described include the steps of labeling myelin in vivo in theanimal's brain tissue with a radioligand described herein, thendetecting a distribution of the radioligand in the animal's braintissue, and then correlating the distribution of the radioligand asdetected in the animal's brain with the efficacy of the remyelinationtherapy. It is contemplated that the labeling step can occur before,during, and after the course of a therapeutic regimen in order todetermine the efficacy of the therapeutic regimen. One way to assess theefficacy of a remyelination therapy is to compare the distribution ofthe radioligand before remyelination therapy with the distribution ofthe radioligand after remyelination therapy has commenced or concluded.

Remyelination therapy as used herein refers to any therapy leading to areduction in severity and/or frequency of symptoms, elimination ofsymptoms and/or underlying cause, prevention of the occurrence ofsymptoms and/or their underlying cause, and improvement or remediationof damage related to demyelination. For example, a remyelination therapycan include administration of a therapeutic agent, therapies for thepromotion of endogenous myelin repair, or a cell based therapy (e.g., astem-cell based therapy).

In another embodiment, methods are provided for screening for amyelination response in an animal's brain tissue to an agent. The methodincludes the initial step of administering an agent to the animal.Myelin in the animal's brain tissue is labeled in vivo with aradioligand as described herein. A distribution of the radioligand inthe animal's brain tissue is then detected using a PET imaging. Finally,the distribution of the radioligand with the myelination response in theanimal's brain tissue is correlated to the agent. One way to assess themyelination response in the animal's brain tissue is to compare thedistribution of the radioligand in an animal's brain tissue, which hasbeen treated with a suspect agent with the distribution of theradioligand in the brain tissue of a control population. “ControlPopulation” as used herein is defined as a population or a tissue samplenot exposed to the agent under study but otherwise as close in allcharacteristics to the exposed group as possible.

The radioligands described herein can also be used to determine if anagent of interest has the potential to modulate demyelination,remyelination, or dysmyelination of axonal regions of an experimentalanimal's brain tissue.

Example 1

This Example describes the design, synthesis, radiolabeling, andmicroPET imaging studies of a series of fluorescent trans-stilbeneradioligands.

Methods

All chemicals and reagents were used as received without furtherpurification. Glassware was dried in an oven at 130° C. and purged witha dry atmosphere prior to use. Unless otherwise mentioned, reactionswere performed open to air. Reactions were monitored by TLC andvisualized by a dual short/long wavelength UV lamp. Flash columnchromatography was performed using 230-400 mesh silica gel (Fisher). NMRspectra were recorded on a Varian Inova 400 spectrometer and a 500 MHzBruker Ascend Avance III HD at room temperature. Chemical shifts for ¹Hand ¹³C NMR were reported as 6, part per million (ppm), and referencedto an internal deuterated solvent central line. Multiplicity andcoupling constants (J) were calculated automatically on MestReNova 10.0,a NMR processing software from Mestrelab Research. The purity of thenewly synthesized compounds as determined by analytical HPLC was >95% onC-18 reversed-phase HPLC (Phenomenex, 10×250 mm),eluent:acetonitrile:H₂O=60:40, flow rate of 3.0 mL/min. HRMS-ESI massspectra were acquired on an Agilent Q-TOF. Fluorescence was measuredwith a Cary Eclipse spectrophotometer using 1×1 cm quartz cuvette in a10 mM acetonitrile solution.

General Method for Synthesis

3-Hydroxy-4-nitrobenzaldehyde (1) or 4-hydroxy-3-nitrobenzaldehyde (2)(1.5 g, 8.9 mmol) was deprotonated with K₂CO₃, (2.48 g, 17.9 mmol) in 20mL DMF followed by dropwise adding alkylene glycol ditosylate (a-b, 1equiv) dissolved in 10 mL DMF at 80° C. The reaction mixtures wereheated with continuous stirring for additional 6 h. After completion ofthe reactions, the mixtures were cooled to room temperature, after 30min, 200 mL ice cold water added, and extracted with 3×50 mL ethylacetate. The organic phases were combined and washed with 20% sodiumbicarbonate (50 mL), 50 mL brine, dried over MgSO₄, and evaporated underreduced pressure. The crude products were purified by flash columnchromatography in an ethyl acetate-hexane mixture.

2-(5-Formyl-2-nitrophenoxy)ethyl 4-Methylbenzenesulfonate (3)

This dull yellow crystalline solid was eluted in 40% EtOAc:hexane, yield1.2 g, (60%). ¹H NMR (400 MHz, chloroform-d) δ 10.07 (s, 1H), 7.95 (d,J=8.1 Hz, 1H), 7.84 (d, J=8.4 Hz, 2H), 7.62 (dd, J=8.1, 1.5 Hz, 1H),7.56 (d, J=1.5 Hz, 2H), 7.41 (dq, J=7.9, 0.7 Hz, 2H), 4.45 (d, J=1.4 Hz,4H), 2.49 (s, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 190.2, 151.6,145.5, 139.7, 132.5, 130.2, 128.2, 126.3, 123.6, 113.9, 67.6, 67.3,21.9.

2-(2-(5-Formyl-2-nitrophenoxy)ethoxy)ethyl 4-Methylbenzene-sulfonate (4)

The off white crystalline compound was eluted in 30% EtOAc:hexane, yield1.0 g, (40%). ¹H NMR (400 MHz, chloroform-d) δ 10.02 (s, 1H), 7.90 (d,J=8.2 Hz, 1H), 7.76 (d, J=8.3 Hz, 2H), 7.59 (d, J=1.5 Hz, 1H), 7.54 (d,J=8.1 Hz, 1H), 7.31 (dt, J=8.0, 0.7 Hz, 1H), 4.32-4.21 (m, 2H),4.20-4.11 (m, 2H), 3.87-3.80 (m, 2H), 3.78-3.71 (m, 2H), 2.41 (s, 3H).¹³C NMR (101 MHz, chloroform-d) δ 190.5, 152.5, 145.1, 139.8, 133.1,130.0, 128.1, 126.1, 122.9, 114.5, 70.1, 69.5, 69.4, 21.9.

3-(2-Fluoroethoxy)-4-nitrobenzaldehyde (5)

3-Hydroxy-4-nitro-benzaldehyde (1, 1.0 g, 6.0 mmol) was deprotonatedwith K₂CO₃ (1.65 g, 12.0 mmol) in 10 mL dry DMF followed by reactionwith 1-fluoro-2-iodoethane (0.8 mL, 9.0 mmol, 2.14 g/mL) at 80° C. Thereaction was monitored by TLC, and after 6 h no starting material wasdetected. The reaction mixture was cooled to room temperature, 100 mLice cold water was added, and the compound was precipitated out,filtered under vacuum, and washed with water and diethyl ether. A yellowamorphous compound was obtained [yield 0.90 g, (70%)] and used withoutfurther purification for the next reaction. ¹H NMR (400 MHz,chloroform-d) δ 10.05 (s, 1H), 7.94 (d, J=7.6 Hz, 1H), 7.69-7.54 (m,2H), 4.82 (d, J=47.5 Hz, 2H), 4.45 (d, J=27.0 Hz, 2H,). ¹³C NMR (101MHz, CDCl₃) δ 190.3, 139.8, 126.7 (2C), 123.6 (2C), 114.0, 81.4 (d,J_(C—F)=173 Hz), 69.4 (d, J_(C—F)=21 Hz). ¹⁹F NMR (376 MHz, CDCl₃) δ−90.90-−91.16 (C—F coupling) −100.01 (F-decoupling).

2-(4-Formyl-2-nitrophenoxy)ethyl 4-Methylbenzenesulfonate (6)

The white crystalline compound was eluted in 40% EtOAc:hexane, yield0.45 g (65%). ¹H NMR (400 MHz, chloroform-d) δ 9.93 (s, 1H), 8.32 (d,J=2.1 Hz, 1H), 8.01 (dd, J=8.8, 2.4 1H), 7.78 (d, J=8.3 Hz, 2H), 7.32(dd, J=8.4, 0.8 Hz, 2H), 7.18 (d, J=8.7 Hz, 1H), 4.57-4.30 (m, 4H), 2.45(s, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 188.8, 155.6, 145.6, 134.9,132.3, 130.3, 129.8, 128.1, 127.5, 114.9, 67.7, 67.2, 21.9.

4-(2-Fluoroethoxy)-3-nitrobenzaldehyde (7)

The off white amorphous compound was precipitated out and was usedwithout further purification, yield 1.1 g (86%). ¹H NMR (400 MHz,chloroform-d) δ 9.95 (s, 1H), 8.36 (dd, J=2.1, 0.8 Hz, 1H), 8.08 (ddd,J=8.7, 2.0, 0.6 Hz, 1H), 7.27-7.25 (m, 1H), 4.94-4.86 (m, 1H), 4.81-4.73(m, 1H), 4.55-4.40 (m, 2H). ¹³C NMR (101 MHz, chloroform-d) δ 188.9,156.2, 134.8, 129.8, 127.7, 115.0, 82.2, 80.4, 69.5, 69.3. ¹⁹F NMR (376MHz, chloroform-d) δ −90-−91.13 (m), −100.01 (s).

tert-Butyl (4-((Diethoxyphosphoryl)methyl)phenyl)carbamate (9)

Compound 9 was synthesized according to our earlier published method. Toa 100 mL round-bottom flask with a magnetic stir bar, diethyl4-aminobenzylphosphonate (8, 5.0 g, 20.56 mmol), di-tert-butyldicarbonate (4.50 g, 21.0 mmol), THF (25 mL), and water (10 mL) wereadded. The reaction was stirred at room temperature open to airovernight. After completion of the reaction, THF was evaporated undervacuum, and the resulting residue was diluted with water and extractedwith ethyl acetate (50 mL×3) three times. The organic layers werecombined and washed twice with water (50 mL×2) and once with brine (50mL). The organic layer was dried over MgSO₄, filtered, and evaporatedunder reduced pressure. The resulting white amorphous compound wasobtained [yield 6.5 g (95%)] and used without further purification. ¹HNMR (400 MHz, chloroform-d) δ 7.30 (br d, J=8.4 Hz, 2H), 7.17-7.19 (m,2H), 6.87 (br s, 1H), 4.04-3.91 (m, 4H), 3.07 (d, ²J_(H, P)=21.2 Hz,2H), 1.49 (s, 9H), 1.22 (td, ³J_(H, H)=6.8 Hz, ⁴J_(H, P)=0.4 Hz, 6H).¹³C NMR (100 MHz, chloroform-d) δ 152.8, 137.4, 130.1, 125.5, 118.4,80.3, 62.0, 32.9 (d, ¹J_(c,p)=138 Hz, 1C), 28.8, 16.3.

General Method for Alkylation of tert-Butyl(4-((Diethoxyphosphoryl)methyl)phenyl)carbamate (10-14)

To an oven-dried 100 mL round-bottom flask purged with argon gas andfitted with a magnetic stirrer were added sodium hydride (0.150 g, 5.82g, 95%) and tert-butyl (4-((diethoxyphosphoryl)methyl)phenyl)-carbamate(9, 1.0 g, 2.9 mmol). The mixture was purged with argon gas, and dry THF(25 mL) at 0° C. was added. Iodo-alkane (4.0 equiv) was added dropwiseafter 30 min at 0° C. under argon gas. The reaction was stirred underargon and allowed to reach room temperature overnight. After completion,the reaction was quenched with water, and THF was removed by vacuum. Theresidue was dissolved in dichloromethane (DCM) and water, and theaqueous layer was extracted three times with DCM (30 mL×3). The organiclayers were combined and washed twice with water (50 mL×2) and once withbrine (50 mL). The organic layer was dried over MgSO₄ and then filteredand concentrated to yield the desired products 10-14 as sticky oil. Thiswas purified over a silica flash column using hexane:ethyl acetate aseluent as required with derivatives.

tert-Butyl (4-((Diethoxyphosphoryl)methyl)phenyl)(methyl)-carbamate (10)

This dark yellow sticky oil was used without further purification, yield0.80 g, (76%). ¹H NMR (400 MHz, chloroform-d) δ 7.24-7.25 (m, 2H), 7.16(d, J=8.0 Hz, 2H), 4.07-3.94 (m, 4H), 3.22 (s, 3H), 3.11 (d,³J_(H, P)=21.6 Hz, 2H), 1.42 (s, 9H), 1.23 (t, J=7.2, 6H). ¹³C NMR (100MHz, chloroform-d) δ 154.5, 142.4, 129.7, 128.4, 125.4, 80.1, 61.9,37.1, 33.0 (d, ²J_(C,P)=138 Hz, 1C), 28.1, 16.3.

tert-Butyl (4-((diethoxyphosphoryl)methyl)phenyl)(ethyl)-carbamate (11)

This clear sticky oil was eluted in 30% EtOAc:hexane, yield 0.65 g(40%). ¹H NMR (400 MHz, chloro-form-d) δ 7.28-7.23 (m, 2H), 7.11 (d,J=8.0 Hz, 2H), 4.00 (dqd, J=8.3, 7.1, 3.7 Hz, 4H), 3.64 (q, J=7.1 Hz,2H), 3.13 (d, J_(H,P)=21.6 Hz, 2H), 1.40 (s, 9H), 1.23 (td, J=7.1, 0.4Hz, 6H), 1.11 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, chloroform-d) δ141.0, 129.9, 129.8, 129.0, 128.9, 126.9, 79.7, 62.0, 61.9, 44.6, 33.1(d, J_(c,p)=138 Hz, 1C), 28.1, 16.2, 16.2, 13.6.

tert-Butyl (4-((Diethoxyphosphoryl)methyl)phenyl)(propyl)-carbamate (12)

This clear sticky oil was eluted in 30% EtOAc:hexane, yield 0.400 g,(25%). ¹H NMR (400 MHz, chloroform-d) δ 7.23 (d, J=2.5 Hz, 1H), 7.21 (d,J=2.6 Hz, 1H), 7.08 (d, J=8.0 Hz, 2H), 3.97 (dqd, J=8.3, 7.1, 3.9 Hz,4H), 3.57-3.47 (m, 2H), 3.10 (d, J_(H,P)=21.6 Hz, 2H), 1.54-1.45 (m,2H), 1.37 (s, 9H), 1.22-1.17 (m, 6H), 0.83 (t, J=7.4 Hz, 3H). ¹³C NMR(101 MHz, chloroform-d) δ 141.3, 141.2, 130.0, 129.9, 128.0, 127.1,127.0, 127.0, 79.8, 62.1, 62.0, 51.5, 33.2 (d, J_(C,P)=138 Hz, 1C),28.2, 21.5, 16.3, 16.2, 11.0.

tert-Butyl (4-((Diethoxyphosphoryl)methyl)phenyl)(isopropyl)-carbamate(13)

This yellow Oil was eluted in 25% EtOAc:hexane, yield 0.41 g (25%). ¹HNMR (400 MHz, chloroform-d) δ 7.24 (dd, J=7.8, 2.0 Hz, 2H), 6.97 (d,J=8.0 Hz, 2H), 4.56-4.41 (m, 1H), 4.02-3.89 (m, 4H), 3.12 (d,J_(H,P)=21.6 Hz, 2H), 1.31 (s, 9H), 1.18 (td, J=7.1, 0.6 Hz, 6H), 1.03(dd, J=6.8, 0.8 Hz, 6H). ¹³C NMR (101 MHz, chloroform-d) δ 130.1, 130.0,129.8, 129.8, 129.7, 79.4, 62.0, 62.0, 33.2 (d, J_(C,P)=138 Hz, 1C),28.1, 21.2, 16.2, 16.2.

tert-Butyl sec-Butyl(4-((diethoxyphosphoryl)methyl)phenyl)-carbamate(14)

This clear oil was eluted in 30% EtOAc:hexane, yield 0.20 g (15%). ¹HNMR (400 MHz, chloroform-d) δ 7.29-7.25 (m, 2H), 7.08-6.98 (m, 2H), 4.21(d, J=10.3 Hz, 1H), 4.06-3.92 (m, 4H), 3.14 (d, J_(H,P)=21.6 Hz, 2H),1.54 (dt, J=13.5, 7.5 Hz, 1H), 1.35 (s, 9H), 1.28 (d, J=7.1 Hz, 1H),1.26-1.17 (m, 6H), 1.05 (d, J=6.9 Hz, 3H), 0.95 (t, J=7.4 Hz, 3H). ¹³CNMR (101 MHz, CDCl₃) δ 154.9, 138.1, 129.9, 129.8, 129.7, 129.6, 129.6,129.5, 129.5, 79.3, 62.0, 61.9, 54.3, 33.2 (d, J_(C,P)=138 Hz, 1C),28.2, 28.1, 19.1, 16.2, 16.1, 11.2.

General Method for the Synthesis of 16-22 (Scheme 3)

To an oven-dried 100 mL round-bottom flask purged with argon and fittedwith a magnetic stirrer was added sodium hydride (2.0 eq., 60%dispersion in mineral oil). The flask was purged with argon, and dry DMF2.0 mL was added. The compound (10-15, 1.1 equiv) was dissolved in dryDMF (2.0 mL) and transferred via syringe in a NaHDMF mixture to a flask.The mixture was stirred under argon at 0° C. for 1 h. 3 or 6 (1.0 equiv)was dissolved in dry DMF (2 mL) and added to the reaction mixture viasyringe under argon at 0° C. The reaction was stirred for 2 h at 0° C.in the dark under argon. The reaction was quenched with water (25 mL)and extracted with ethyl acetate (30 mL×3) three times. The organiclayers were combined and washed with water (25 mL×2) twice and once withbrine (25 mL). The organic layer was dried over MgSO₄, filtered, andconcentrated to give sticky oil as crude product. This was purified bydissolving in a minimal amount of DCM and made slurry with silica geland dried under vacuum. The compound was eluted in 10-15% ethylacetate:hexane in a flash column.

(E)-2-(5-(4-((tert-Butoxycarbonyl)(methyl)amino)styryl)-2-nitrophenoxy)ethyl-4-methylbenzenesulfonate(16)

This yellow amorphous compound was eluted in 15% EtOAc:hexane, yield0.140 g (40%). ¹H NMR (400 MHz, chloroform-d) δ 7.88 (d, J=8.5 Hz, 1H),7.84-7.80 (m, 2H), 7.50 (d, J=8.5 Hz, 2H), 7.37-7.33 (m, 2H), 7.29 (d,J=8.4 Hz, 2H), 7.22-7.19 (m, 1H), 7.18-7.16 (m, 1H), 7.11 (d, J=1.7 Hz,1H), 6.99 (d, J=16.3 Hz, 1H), 4.41 (q, J=2.1 Hz, 4H), 3.29 (s, 3H), 2.44(s, 3H), 1.48 (s, 9H). ¹³C NMR (101 MHz, chloroform-d) δ 145.4, 144.5,144.2, 132.8, 130.2, 128.2, 127.4, 126.7, 125.9, 125.6, 119.4, 113.0,67.8, 37.3, 28.5, 21.9.

(E)-2-(5-(4-((tert-Butoxycarbonyl)(ethyl)amino)styryl)-2-nitrophenoxy)ethyl-4-methylbenzenesulfonate(17)

The yellow sticky solid product was eluted in a silica flash column with15% EtOAc:hexane. The yellow amorphous product yield 0.125 (40%). ¹H NMR(400 MHz, chloroform-d) δ 7.87 (d, J=8.5 Hz, 1H), 7.84-7.80 (m, 2H),7.50 (d, J=8.5 Hz, 2H), 7.38-7.33 (m, 2H), 7.24 (d, J=8.5 Hz, 2H),7.22-7.19 (m, 1H), 7.18-7.16 (m, 1H), 7.11 (d, J=1.7 Hz, 1H), 7.00 (d,J=16.3 Hz, 1H), 4.48-4.35 (m, 4H), 3.70 (q, J=7.1 Hz, 2H), 2.44 (s, 3H),1.46 (s, 9H), 1.17 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, chloroform-d) δ152.3, 145.4, 144.1, 143.1, 133.5, 132.7, 132.5, 130.1, 128.1, 127.4,127.1, 126.7, 126.0, 119.3, 112.9, 80.50, 67.7, 67.6, 45.0, 28.5, 21.8,14.1.

(E)-2-(5-(4-((tert-Butoxycarbonyl)(propyl)amino)styryl)-2-nitrophenoxy)ethyl-4-methylbenzenesulfonate(18)

The yellow crystalline compound was eluted on a silica flash column in15% EtOAc:hexane. Yield 0.165 g (35%). ¹H NMR (400 MHz, chloroform) δ7.87 (d, J=8.5 Hz, 1H), 7.85-7.79 (m, 2H), 7.50 (d, J=8.4 Hz, 2H), 7.35(dt, J=7.9, 0.7 Hz, 2H), 7.23 (d, J=8.4 Hz, 2H), 7.21-7.19 (m, 1H),7.18-7.16 (m, 1H), 7.11 (d, J=1.7 Hz, 1H), 7.00 (d, J=16.2 Hz, 1H),4.46-4.37 (m, 4H), 3.67-3.57 (m, 2H), 2.44 (s, 3H), 1.60 (s, 1H), 1.57(s, 1H), 1.45 (s, 9H), 0.89 (t, J=7.4 Hz, 3H). ¹³C NMR (101 MHz,chloroform-d) δ 152.3, 145.4, 144.1, 133.5, 132.7, 132.5, 130.1, 128.1,127.4, 127.3, 126.7, 126.0, 119.3, 112.9, 80.5, 67.7, 67.6, 51.6, 28.5,21.9, 21.8, 11.3.

(E)-2-(5-(4-((tert-Butoxycarbonyl)(isopropyl)amino)styryl)-2-nitrophenoxy)ethyl-4-methylbenzenesulfonate(19)

The yellow amorphous compound was eluted on a silica flash column in 15%EtOAc:hexane, yield 0.065 g (30%). ¹H NMR (500 MHz, chloroform) δ 7.87(dd, J=8.5, 2.0 Hz, 1H), 7.84-7.79 (m, 2H), 7.53-7.48 (m, 2H), 7.35 (d,J=7.8 Hz, 2H), 7.23-7.17 (m, 2H), 7.14-7.09 (m, 3H), 7.06-7.00 (m, 1H),4.57-4.46 (m, 1H), 4.42 (h, J=4.3, 3.7 Hz, 4H), 2.44 (s, 3H), 1.39 (s,9H), 1.13 (dd, J=6.9, 2.0 Hz, 6H). ¹³C NMR (126 MHz, chloroform-d) δ154.9, 152.4, 145.4, 144.1, 140.2, 138.8, 134.8, 132.8, 132.6, 130.5,130.2, 128.2, 127.3, 126.7, 126.5, 119.5, 113.1, 80.1, 67.8, 67.8, 28.6,21.9, 21.8.

(E)-2-(5-(4-((tert-Butoxycarbonyl)(sec-butyl)amino)styryl)-2-nitrophenoxy)ethyl-4-methylbenzenesulfonate(20)

The yellow sticky solid was eluted in 10% EtOAc:hexane, yield 0.040 g(15%). 1H NMR (400 MHz, chloroform-d) δ 7.88 (d, J=8.5 Hz, 1H),7.85-7.80 (m, 2H), 7.53-7.49 (m, 2H), 7.37-7.33 (m, 2H), 7.23-7.20 (m,1H), 7.18 (d, J=1.9 Hz, 1H), 7.15-7.10 (m, 3H), 7.02 (d, J=16.3 Hz, 1H),4.45-4.38 (m, 4H), 4.23 (d, J=7.0 Hz, 1H), 2.44 (s, 3H), 1.67-1.58 (m,1H), 1.40 (s, 9H), 1.37-1.32 (m, 1H), 1.12 (d, J=6.8 Hz, 3H), 0.98 (t,J=7.4 Hz, 3H). 13C NMR (101 MHz, chloroform-d) δ 152.3, 145.4, 144.0,134.6, 132.7, 132.5, 130.2, 130.1, 130.1, 128.1, 127.2, 126.7, 126.4,119.4, 113.0, 80.1, 67.7, 67.7, 66.8, 55.2, 28.7, 28.5, 21.8, 19.7,11.6.

(E)-2-(2-Nitro-5-(4-nitrostyryl)phenoxy)ethyl-4-methylbenzenesulfonate(21)

A yellow amorphous precipitate formed after quenching with water and wasthen washed several times with water and diethyl ether. The yellowamorphous product yield was 0.150 g (75%). ¹H NMR (400 MHz, DMSO-d6) δ8.31-8.25 (m, 2H), 7.93 (d, J=8.4 Hz, 1H), 7.91-7.86 (m, 2H), 7.79-7.74(m, 2H), 7.65 (d, J=16.5 Hz, 1H), 7.60-7.52 (m, 2H), 7.42 (t, J=8.2 Hz,3H), 4.50-4.34 (m, 4H), 2.39 (s, 3H). ¹³C NMR (101 MHz, DMSO) δ 151.7,147.4, 145.7, 143.7, 143.2, 139.1, 132.6, 131.7, 131.1, 130.8, 128.5,128.4, 128.2, 128.1, 126.5, 126.5, 124.8, 124.8, 124.7, 120.4, 113.6,69.2, 67.6, 21.8.

(E)-2-(5-(4-((tert-Butoxycarbonyl)(methyl)amino)styryl)-2-nitrophenoxy)ethyl-4-methylbenzenesulfonate(22)

The yellow crystalline compound was purified over a silica flash columnin 20% EtOAc:hexane, yield 0.240 g (50%). ¹H NMR (500 MHz, chloroformd)δ 7.96 (s, 1H), 7.82 (d, J=8.1 Hz, 2H), 7.62 (dd, J=8.6, 2.2 Hz, 1H),7.46 (d, J=8.3 Hz, 2H), 7.37 (d, J=8.1 Hz, 2H), 7.27 (s, 2H), 7.07-7.02(m, 2H), 6.97 (d, J=16.3 Hz, 1H), 4.45-4.33 (m, 4H), 3.29 (s, 3H), 2.46(s, 3H), 1.49 (s, 9H). ¹³C NMR (126 MHz, chloroform-d) δ 154.5, 150.3,145.1, 143.6, 133.2, 132.3, 131.5, 129.9, 129.3, 127.8, 126.6, 125.4,125.0, 122.9, 115.3, 80.5, 67.4, 67.4, 37.0, 28.2, 21.6.

Synthesis of(E)-2-(2-(2-Nitro-5-(4-nitrostyryl)phenoxy)ethoxy)-ethyl-4-methylbenzene-sulfonate23 (Scheme 4)

To an oven-dried 100 mL round-bottom flask purged with argon and fittedwith a magnetic stirring bar was added sodium hydride (0.80 g, 2.0 mmol,60% dispersion in mineral oil). The flask was purged with argon, and dryDMF 2.0 mL was added. The diethyl (4-nitrobenzyl) phosphonate (15, 0.30g, 1.1 mmol) was dissolved in dry DMF (2.0 mL), and the NaH-DMF mixturewas transferred via syringe to a flask. The mixture was then stirredunder argon at 0° C. for 1 h. The tosylated 4-nitrobenzaldehyde (4,0.360 g 1.0 mmol) was dissolved in dry DMF (2 mL) and added to thereaction mixture via syringe under argon at 0° C. The reaction wasstirred again for 2 h at 0° C. in the dark under argon. The reaction wasquenched with 25 mL of ice cold water, and the compound precipitatedout. It was then filtered under vacuum, washed with water several timesfollowed by diethyl ether, and used for the next step without furtherpurification. The yellow amorphous compound was dried under high vacuum,yield 0.390 g (80%). ¹H NMR (400 MHz, DMSO-d6) δ 8.31-8.25 (m, 2H), 7.95(d, J=8.4 Hz, 1H), 7.92-7.87 (m, 2H), 7.79-7.74 (m, 2H), 7.68 (d, J=16.5Hz, 1H), 7.64-7.55 (m, 2H), 7.42 (dd, J=9.1, 3.0 Hz, 3H), 4.30 (t, J=4.6Hz, 2H), 4.19-4.09 (m, 2H), 3.79-3.62 (m, 4H), 2.37 (s, 3H). ¹³C NMR(101 MHz, DMSO) δ 151.8, 146.7, 144.8, 143.1, 142.6, 138.5, 132.3,131.2, 130.4, 130.1, 127.8, 127.6, 125.9, 124.2, 119.4, 113.1, 70.0,69.1, 68.5, 68.2, 21.1.

(E)-2-(2-(2-Fluoroethoxy)ethoxy)-1-nitro-4-(4-nitrostyryl)benzene (24)

Compound (23, 0.15 g, 0.28 mmol) and Kryptofix 2.2.2. (K₂₂₂, 0.33 g,0.85 mmol) with CsF (0.09 g, 0.57 mmol) as the source of fluoride-19were mixed together in a 100 mL round-bottom flask in dry acetonitrile(20 mL), and the reaction was purged with argon. The reaction mixturewas heated at 80° C. for 4 h and monitored by TLC. Once the reaction wascomplete, acetonitrile was evaporated under reduced pressure, and thereaction was quenched with ice cold water. The dark brown precipitatewas filtered under vacuum and washed with water several times anddiethyl ether once. The light brown amorphous product yield was 0.090 g,and 84% was used with further purification. ¹H NMR (400 MHz,chloroform-d) δ 8.26 (d, J=8.3 Hz, 2H), 7.92 (d, J=8.0 Hz, 1H), 7.68 (d,J=8.5 Hz, 2H), 7.29 (d, J=1.6 Hz, 1H), 7.25-7.18 (m, 3H), 4.60 (dt,J=47.8, 4.0 Hz, 2H), 4.37 (t, J=4.7 Hz, 2H), 3.99 (t, J=4.6 Hz, 2H),3.88 (dt, J=30.1, 3.8 Hz, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 170.4,156.3, 153.2, 136.1, 135.6, 134.5, 131.1, 131.1, 131.0, 130.5, 127.7,127.6, 126.7, 124.5, 119.4, 117.5, 113.6, 105.3, 94.6, 86.6 (d, JC—F=164Hz), 70.1 (JC—F, J=43 Hz). 19F NMR (376 MHz, chloroform-d) δ −100.01.

(E)-4-(4-Aminostyryl)-2-(2-(2-fluoroethoxy)ethoxy)aniline (25)

To a 100 mL round-bottom flask with a magnetic stirring bar was addedtin(II) chloride (0.285 g, 1.5 mmol).(E)-2-(2-(2-fluoroethoxy)-ethoxy)-1-nitro-4-(4-nitrostyryl)benzene (24,0.050 g, 0.15 mmol) was added to the tin(II) chloride solution of ethylacetate (15 mL) and ethanol (10 mL). The reaction mixture was refluxedunder a water-cooled condenser in an oil bath at 70° C. and stirredovernight open to air. The reaction was monitored via TLC and aftercompletion of the reaction was cooled to room temperature, and thesolvent was removed by vacuum. The compound was dissolved in aq Na₂CO₃(20%) until bubbles stopped forming and was washed with ethyl acetate 3times (30 mL×3), water (50 mL×2), followed by brine (50 mL). The organiclayer was dried over the MgSO₄, filtered, and concentrated to give thecrude 25. The crude product was dissolved in minimal DCM and purified onsilica by flash column chromatography with a mobile phase ofEtOAc:hexane, and 25 was eluted in 10-15% EtOAc:hexane yielding a stickybrown solid, yield 0.02 g, (49%). ¹H NMR (400 MHz, chloroform-d) δ7.28-7.24 (m, 2H), 6.95 (d, J=1.8 Hz, 1H), 6.90 (dd, J=8.0, 1.8 Hz, 1H),6.78 (s, 2H), 6.66-6.63 (m, 2H), 6.62 (d, J=1.9 Hz, 1H), 4.68-4.61 (m,1H), 4.57-4.49 (m, 1H), 4.27-4.15 (m, 2H), 3.90-3.86 (m, 2H), 3.86-3.80(m, 2H), 3.79-3.72 (m, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 146.2,145.3, 136.1, 128.61, 128.4, 127.1, 125.3, 125.0, 120.4, 115.1, 115.0,110.1, 81.8 (d, JC—F=171 Hz), 69.4 (d, JC—F=21.0 Hz). 19F NMR (376 MHz,chloroform-d) δ −89.8-−90.7 (m), −100.0.

General Method for the Synthesis of 26-31 (Scheme 5, Step (i))

To an oven-dried 100 mL round-bottom flask with a magnetic stirring barwas added sodium hydride (NaH, 2.0 equiv, 60%). The flask was purgedwith argon, and 2.0 mL dry DMF was added. The solution was stirred underargon in an ice bath (0° C.). Boc-N-R1-diethyl benzoylphosphonate anddiethyl (4-nitrobenzyl)phosphonate (10-15, 1.2 equiv) in 2.0 mL dry DMFwere added to the solution of NaH. This sodium hydride and intermediate(10-15) mixture was stirred at 0° C. under argon for 1 h. Then 2.0 mLdry DMF solution of fluorinated nitrobenzaldehyde was transferred viasyringe to the reaction mixture under argon at 0° C. The reaction wascontinued for another 2 h at 0° C. under argon and monitored via TLC.Once completed, the reaction was quenched with ice cold water (50 mL)and extracted with ethyl acetate (50 mL×3) three times. The organiclayer was washed twice with water (50 mL×2) and once with brine (50 mL).The organic layer was dried over MgSO₄, filtered, and concentrated togive a yellow crude solid/oil. The solid was dissolved in 2 mL ethylacetate and loaded onto a silica column and eluted with ethyl acetateand hexane.

tert-Butyl(E)-(4-(3-(2-Fluoroethoxy)-4-nitrostyryl)phenyl)-(methyl)carbamate (26)

This yellow amorphous compound was purified on a silica flash column in15% EtOAc:hexane, yield 0.080 g, (30%). ¹H NMR (400 MHz, chloroform-d) δ7.91-7.86 (m, 1H), 7.47 (d, J=8.1 Hz, 2H), 7.27 (d, J=8.1 Hz, 2H), 7.19(d, J=7.2 Hz, 1H), 7.14 (d, J=2.9 Hz, 2H), 7.00 (d, J=16.4 Hz, 1H),4.91-4.85 (m, 1H), 4.80-4.71 (m, 1H), 4.41 (dt, J=26.9, 3.7 Hz, 2H),3.27 (s, 3H), 1.46 (s, 9H). ¹³C NMR (101 MHz, chloroform-d) δ 152.8,144.4, 144.1, 133.0, 132.8, 132.6, 128.0, 127.3, 126.8, 126.7, 126.1,125.6, 119.2, 113.2, 81.8 (d, J_(C—F)=171 Hz), 69.4 (d, J_(C—F)=21 Hz),37.3, 28.5. ¹⁹F NMR (376 MHz, chloroform-d) δ −91.0 (m), −100.01 (s).

tert-Butyl(E)-Ethyl(4-(3-(2-fluoroethoxy)-4-nitrostyryl)phenyl)-carbamate (27)

The yellow sticky solid was purified on a silica flash column in 15%EtOAc:hexane, yield 0.110 g, (70%). ¹H NMR (400 MHz, chloroform-d) δ7.91 (d, J=8.4 Hz, 1H), 7.50 (d, J=8.4 Hz, 2H), 7.24 (s, 1H), 7.24-7.21(m, 2H), 7.21-7.19 (m, 1H), 7.18-7.16 (m, 2H), 7.02 (d, J=16.3 Hz, 1H),4.93-4.87 (m, 1H), 4.82-4.76 (m, 1H), 4.48-4.38 (m, 2H), 3.70 (q, J=7.1Hz, 2H), 1.46 (s, 9H), 1.17 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz,chloroform-d) δ 152.7, 144.0, 143.1, 133.6, 132.5, 127.4, 127.1, 126.7,126.2, 119.1, 113.2, 81.8 (d, J_(C—F)=171 Hz), 69.4 (d, J_(C—F)=21 Hz),45.0, 28.5, 14.1. ¹⁹F NMR (376 MHz, chloroform-d) δ −91.0 (m), −100.01(s).

tert-Butyl(E)-(4-(3-(2-Fluoroethoxy)-4-nitrostyryl)phenyl)-(propyl)carbamate (28)

This yellow amorphous compound was purified on a silica flash column in15% EtOAc:hexane, yield 0.150 g, (50%). ¹H NMR (400 MHz, chloroform-d) δ7.89 (d, J=8.5 Hz, 1H), 7.48 (d, J=8.5 Hz, 2H), 7.22 (s, 1H), 7.21-7.18(m, 2H), 7.16-7.13 (m, 2H), 7.00 (d, J=16.3 Hz, 1H), 4.91-4.86 (m, 1H),4.79-4.74 (m, 1H), 4.47-4.43 (m, 1H), 4.40-4.36 (m, 1H), 3.63-3.57 (m,2H), 1.57 (d, J=7.4 Hz, 1H), 1.53 (d, J=5.8 Hz, 1H), 1.43 (s, 9H), 0.87(t, J=7.4 Hz, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 152.8, 144.0,143.3, 133.6, 132.6, 127.4, 127.3, 126.7, 126.3, 119.2, 113.3, 81.8 (d,J_(C—F)=171 Hz), 69.4 (d, J_(C—F)=21 Hz), 51.7, 28.5, 22.0, 11.4. ¹⁹FNMR (376 MHz, chloroform-d) δ −91.0 (m), −100.01 (s).

tert-Butyl(E)-(4-(3-(2-Fluoroethoxy)-4-nitrostyryl)phenyl)-(isopropyl)carbamate(29)

This yellow crystalline compound was purified on a silica flash columnin 15% EtOAc:hexane, yield 0.165 g, (40.0%). ¹H NMR (400 MHz,chloroform-d) δ 7.91 (d, J=8.4 Hz, 1H), 7.51 (d, J=8.3 Hz, 2H),7.24-7.20 (m, 1H), 7.19-7.17 (m, 2H), 7.14-7.09 (m, 2H), 7.05 (d, J=16.3Hz, 1H), 4.93-4.86 (m, 1H), 4.78 (d, J=4.1 Hz, 1H), 4.57-4.49 (m, 1H),4.48-4.37 (m, 2H), 1.39 (s, 9H), 1.13 (d, J=6.8 Hz, 6H). ¹³C NMR (101MHz, chloroform-d) δ 152.8, 143.9, 134.8, 132.6, 130.5, 127.3, 126.7,126.7, 119.2, 113.3, 81.8 (d, J_(C—F)=171 Hz), 69.4 (d, J_(C—F)=21 Hz),48.9, 28.6, 21.8. ¹⁹F NMR (376 MHz, chloroform-d) δ −91.0 (m), −100.01(s).

tert-Butyl(E)-sec-Butyl(4-(3-(2-fluoroethoxy)-4-nitrostyryl)-phenyl)carbamate (30)

This yellow crystalline, compound was purified on a silica flash columnin 15% EtOAc:hexane, yield 0.105 g (55%). ¹H NMR (500 MHz, chloroform-d)δ 7.96 (dd, J=8.5, 1.9 Hz, 1H), 7.60-7.52 (m, 2H), 7.35-7.25 (m, 2H),7.24 (s, 1H), 7.21-7.15 (m, 2H), 7.10 (dd, J=16.3, 1.9 Hz, 1H), 4.89(dt, J=47.3, 3.0 Hz, 2H), 4.49 (dt, J=26.9, 3.1 Hz, 2H), 4.28 (h, J=7.3Hz, 1H), 1.67 (dt, J=9.9, 4.8 Hz, 2H), 1.46 (s, 9H), 1.18 (dd, J=6.9,1.9 Hz, 3H), 1.04 (td, J=7.4, 1.8 Hz, 3H). ¹³C NMR (126 MHz,chloroform-d) δ 155.1, 152.7, 143.9, 140.5, 138.9, 134.6, 132.5, 130.1,127.2, 126.6, 126.6, 119.2, 113.3, 81.8 (d, J_(C—F)=171 Hz), 69.4 (d,J_(C—F)=21 Hz), 55.2, 28.7, 28.5, 19.6, 11.6. ¹⁹F NMR (376 MHz,chloroform-d) δ −90.93 (tt, J=47.3, 26.9 Hz), −100.01.

(E)-2-(2-Fluoroethoxy)-1-nitro-4-(4-nitrostyryl)benzene (31)

The yellow amorphous, precipitate was washed with water and diethylether, yield 0.280 g, (80%). ¹H NMR (400 MHz, DMSO-d6) δ 8.29 (d, J=8.8Hz, 2H), 7.96 (d, J=8.5 Hz, 1H), 7.90 (d, J=8.6 Hz, 2H), 7.66 (s, 2H),7.59 (d, J=16.4 Hz, 1H), 7.43 (d, J=8.5 Hz, 1H), 4.80 (d, J=47.4 Hz,2H), 4.52 (d, J=29.8 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 151.5, 143.1,142.6, 131.2, 130.5, 127.8, 125.9, 124.2, 119.7, 113.2, 81.8 (d,J_(C—F)=171 Hz), 69.4 (d, J_(C—F)=21 Hz). ¹⁹F NMR (376 MHz, DMSO) δ−89.76, −89.84.

General Method for the Boc-Deprotection and Reduction (32-39, Scheme 5,Step (ii))

To a 100 mL round-bottom flask fitted with a stirring bar was addedtin(II) chloride (10 equiv). The compound to be reduced (26-31, 38) wasdissolved in ethyl acetate (25 mL) and ethanol (15 mL) and added totin(II) chloride. The mixture was fitted with a water condenser, heatedto 80° C. for 6 h open to air in the dark. The reaction were monitoredthrough TLC, and after completion, solvent was removed by vacuum. Theresidue was quenched by aq Na₂CO₃ (20%) until bubbles stopped forming.The compound was extracted in ethyl acetate (30 mL×3) three times andwashed with aq Na₂CO₃ (20%) 2 times (30 mL×2) and once with water (50mL) followed by brine (50 mL). It was then dried over Na₂SO₄, andsolvent was removed by reducing the pressure. The crude product wasdissolved in a minimal DCM slurry on silica and purified on silica byflash column chromatography using 10-15% EtOAc:hexane.

(E)-4-(4-Amino-3-(2-fluoroethoxy)styryl)-N-methylaniline (32)

The brown amorphous compound was purified on a silica flash column andeluted with 20% EtOAc:hexane, yield 0.02 g, (55%). ¹H NMR (400 MHz,chloroform-d) δ 7.38 (d, J=8.6 Hz, 2H), 7.00 (dd, J=4.2, 2.4 Hz, 2H),6.87 (d, J=1.9 Hz, 2H), 6.75 (d, J=8.4 Hz, 1H), 6.68-6.60 (m, 2H),4.93-4.75 (m, 2H), 4.40-4.29 (m, 2H), 2.91 (s, 3H). ¹³C NMR (101 MHz,chloroform-d) δ 148.9, 146.3, 129.4, 127.6, 127.5, 125.9, 125.0, 120.9,115.6, 112.8, 112.8, 112.7, 112.7, 110.0, 81.2 (d, J_(C—F)=181 Hz), 68.1(d, J_(C—F)=21 Hz), 30.9. ¹⁹F NMR (376 MHz, chloroform-d) δ−90.53-−91.75 (m), −100.01. HR-MS (ESI) m/z calculated for (C₁₇H₁₉FN₂O)[M+H]⁺ 287.1554, found 287.1550. HPLC purity: 96.26%, retention time10.07 min.

(E)-4-(4-Amino-3-(2-fluoroethoxy)styryl)-N-ethylaniline (33)

The brown amorphous compound was purified on a silica flash column andeluted with 20% EtOAc:hexane, yield 0.045 g (64%). ¹H NMR (400 MHz,chloroform-d) δ 7.34-7.29 (m, 2H), 6.97-6.93 (m, 2H), 6.81 (d, J=1.6 Hz,2H), 6.70 (d, J=8.4 Hz, 1H), 6.63-6.56 (m, 2H), 4.91-4.70 (m, 2H),4.38-4.24 (m, 2H), 3.88 (s, 2H), 3.18 (q, J=7.2 Hz, 2H), 1.26 (t, J=7.1Hz, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 146.6, 145.8, 135.7, 128.9,127.1, 126.7, 125.4, 124.3, 120.4, 115.1, 113.1, 109.5, 81.8 (d,J_(C—F)=171 Hz), 67.7 (d, J_(C—F)=20 Hz), 38.3, 14.7. ¹⁹F NMR (376 MHz,chloroform-d) δ −91.36 (tt, J=47.6, 28.1 Hz), −100.01. HR-MS (ESI) m/zcalculated for (C₁₈H₂₁FN₂O) [M+H]⁺ 301.1711, found 301.1706. HPLCpurity: 100.0%, retention time 13.12 min. C-18 reversed-phase HPLC(Phenomenex, 10×250 mm), eluent:acetonitrile:H₂O=60:40, flow rate of 3.0mL/min.

(E)-4-(4-Amino-3-(2-fluoroethoxy)styryl)-N-propylaniline (34)

The brown amorphous compound was purified on a silica flash column andeluted with 20% EtOAc:hexane, yield 0.070 g, (70%). H NMR (400 MHz,chloroform-d) δ 7.31 (d, J=8.6 Hz, 2H), 6.95 (dt, J=4.2, 2.2 Hz, 2H),6.81 (d, J=2.0 Hz, 2H), 6.70 (d, J=8.4 Hz, 1H), 6.58 (d, J=8.6 Hz, 2H),4.90-4.80 (m, 1H), 4.77-4.69 (m, 1H), 4.38-4.25 (m, 2H), 3.84 (s, 3H),3.11 (t, J=7.1 Hz, 2H), 1.65 (h, J=7.3 Hz, 2H), 1.01 (t, J=7.4 Hz, 3H).¹³C NMR (101 MHz, chloroform-d) δ 147.9, 146.1, 136.0, 129.2, 127.4,127.1, 125.7, 124.7, 120.7, 115.4, 112.9, 109.8, 82.1 (d, J_(C—F)=171Hz, 1C), 68.0 (d, J_(C—F)=20 Hz, 1C), 45.9, 22.8, 11.8. ¹⁹F NMR (376MHz, chloroform-d) δ −91.36 (tt, J=47.7, 28.2 Hz), −100.01. HR-MS (ESI)m/z calculated for (C₁₉H₂₃FN₂O) [M+H]+ 315.1867, found 315.1866. HPLCpurity: 100.0%, retention time 17.37 min. C-18 reversed-phase HPLC(Phenomenex, 10×250 mm), eluent:acetonitrile:H₂O=60:40, flow rate of 3.0ml/min.

(E)-4-(4-Amino-3-(2-fluoroethoxy)styryl)-N-isopropylaniline (35)

The yellow amorphous compound was purified on a silica flash column andeluted with 20% EtOAc:hexane, yield 0.10 g (90%). ¹H NMR (400 MHz,chloroform-d) δ 7.30 (d, J=8.7 Hz, 2H), 6.95 (dq, J=4.3, 1.8 Hz, 2H),6.81 (d, J=1.8 Hz, 2H), 6.69 (d, J=8.4 Hz, 1H), 6.56 (d, J=8.6 Hz, 2H),4.89-4.81 (m, 1H), 4.77-4.72 (m, 1H), 4.41-4.26 (m, 2H), 3.87 (s, 2H),3.65 (p, J=6.3 Hz, 1H), 1.22 (d, J=6.3 Hz, 6H). ¹³C NMR (101 MHz,chloroform-d) δ 146.6, 145.8, 135.7, 128.9, 127.1, 126.7, 125.4, 124.3,120.4, 115.1, 113.1, 109.5, 81.8 (d, JC—F=171 Hz), 67.7 (d, JC—F=20 Hz),44.0, 22.8. ¹⁹F NMR (376 MHz, chloroform-d) δ −91.36 (tt, J=47.6, 28.1Hz), −100.01. HRMS (ESI) m/z calculated for (C₁₉H₂₃FN₂O) [M+H]+315.1867, found 315.1867. HPLC purity: 100.0%, retention time 16.22 min.C-18 reversed-phase HPLC (Phenomenex, 10×250 mm),eluent:acetonitrile:H2O=60:40, flow rate of 3.0 mL/min.

(E)-4-(4-Amino-3-(2-fluoroethoxy)styryl)-N-(sec-butyl)aniline (36)

This brown amorphous compound was purified on a silica flash column andeluted with 20% EtOAc:hexane, yield 0.040 g (70%). ¹H NMR (400 MHz,chloroform-d) δ 7.33-7.27 (m, 2H), 6.95 (dt, J=4.4, 2.0 Hz, 2H), 6.80(d, J=2.5 Hz, 2H), 6.69 (d, J=8.4 Hz, 1H), 6.58-6.53 (m, 2H), 4.88-4.83(m, 1H), 4.77-4.70 (m, 1H), 4.36-4.32 (m, 1H), 4.30-4.24 (m, 1H), 3.82(s, 3H), 3.42 (q, J=6.3 Hz, 1H), 1.69-1.57 (m, 1H), 1.53-1.44 (m, 1H),1.18 (d, J=6.3 Hz, 3H), 0.97 (d, J=7.4 Hz, 3H). ¹³C NMR (101 MHz,chloroform-d) δ 147.1, 146.1, 136.0, 129.3, 127.5, 126.9, 125.8, 124.6,120.7, 115.4, 113.3, 109.9, 82.1 (d, JC—F=171 Hz), 68.1 (d, JC—F=20 Hz),50.0, 29.8, 20.4, 10.5. 19F NMR (376 MHz, chloroform-d) δ −91.39 (tt,J=47.5, 27.9 Hz), −100.01. HR-MS (ESI) m/z calculated for (C₂₀H₂₅FN₂O)[M+H]+ 329.2024, found 329.2020. HPLC purity: 100.0%, retention time23.37 min. C-18 reversed-phase HPLC (Phenomenex, 10×250 mm),eluent:acetonitrile:H₂O=60:40, flow rate of 3.0 mL/min.

(E)-4-(4-Aminostyryl)-2-(2-fluoroethoxy)aniline (37)

This dark red amorphous compound was purified on a silica flash columnand eluted with 20% EtOAc:hexane, yield 0.085 g (64%). ¹H NMR (400 MHz,chloroform-d) δ 7.31 (d, J=8.5 Hz, 2H), 6.97 (dq, J=3.2, 1.8 Hz, 2H),6.84 (s, 2H), 6.71 (d, J=8.5 Hz, 1H), 6.70-6.66 (m, 2H), 4.90-4.71 (m,2H), 4.38-4.25 (m, 2H), 3.82 (s, 4H). ¹³C NMR (101 MHz, chloroform-d) δ146.2, 145.8, 136.3, 129.0, 128.7, 127.5, 125.6, 120.9, 115.5, 109.9,82.2 (d, JC—F=171 Hz), 68.1 (d, JC—F=20 Hz). ¹⁹F NMR (376 MHz,chloroform-d) δ −90.99-−91.82 (m), −100.01. HR-MS (ESI) m/z calculatedfor (C₁₆H₁₇FN₂O) [M+H]+ 273.1398, found 273.1396. HPLC purity: 100.0%,retention time 7.07 min. C-18 reversed-phase HPLC (Phenomenex, 10×250mm), eluent:acetonitrile:H₂O=60:40, flow rate of 3.0 mL/min.

tert-Butyl(E)-(4-(4-(2-Fluoroethoxy)-3-nitrostyryl)phenyl)-(methyl)carbamate (38)

Following the general method of Wittig-Homer reaction as describedabove, compound 38 was synthesized as a yellow sticky solid, which waspurified on silica flash column in 15% EtOAc:hexane, yield 0.265 g(75%). ¹H NMR (400 MHz, chloroformd) δ 7.92 (d, J=2.2 Hz, 1H), 7.58 (dd,J=8.7, 2.3 Hz, 1H), 7.42-7.38 (m, 2H), 7.24-7.18 (m, 2H), 7.04 (d, J=8.7Hz, 1H), 6.94 (d, J=12.2 Hz, 2H), 4.83-4.77 (m, 1H), 4.74-4.67 (m, 1H),4.40-4.28 (m, 2H), 3.24 (s, 3H), 1.43 (s, 9H). ¹³C NMR (101 MHz,chloroform-d) δ 154.3, 150.6, 143.4, 133.2, 131.4, 131.3, 131.0, 129.1,128.9, 127.0, 126.4, 125.2, 125.0, 122.78, 115.3, 81.2 (d, JC—F=181 Hz),69.0 (d, JC—F=21 Hz), 36.9, 28.1. ¹⁹F NMR (376 MHz, chloroform-d) δ−90.96 (tt, J=47.3, 27.1 Hz), −100.01.

(E)-4-(4-(2-Fluoroethoxy)-3-nitrostyryl)-N-methylaniline (39)

Following the general method of Boc-deprotection and reduction asdescribed above, compound 39 was synthesized as an off-white amorphouscompound, which was purified through precipitation out of EtOAc:hexane(2:3), yield 0.050 g (30%). ¹H NMR (500 MHz, chloroform-d) δ 7.33 (d,J=8.0 Hz, 2H), 6.90 (s, 1H), 6.85 (s, 1H), 6.80 (s, 1H), 6.76 (s, 1H),6.67 (s, 1H), 6.59 (d, J=8.1 Hz, 2H), 4.81 (s, 1H), 4.72 (s, 1H), 4.25(d, J=27.1 Hz, 2H), 3.84 (s, 4H), 2.86 (s, 3H). ¹³C NMR (126 MHz, DMSO)δ 149.3, 144.5, 138.0, 131.4, 127.2, 126.3, 125.0, 123.4, 114.9, 112.5,111.7, 110.9, 82.4 (d, JC—F=166 Hz), 67.8 (d, JC—F=19 Hz). 29.6. ¹⁹F NMR(376 MHz, chloroform-d) δ −90.96 (tt, J=47.3, 27.1 Hz), −100.01. HR-MS(ESI) m/z calculated for (C₁₇H₁₉FN₂O) [M+H]+ 287.1554, found 287.1552.HPLC purity: >99%, retention time 10.25 min C-18 reversed-phase HPLC(Phenomenex, 10×250 mm), eluent:acetonitrile:H₂O=60:40, flow rate of 3.0ml/min.

Animal Preparation and Studies

All animal experiments were performed in accordance with guidanceprotocol approved by the Institutional Animal Care and Use Committee(IACUC) of Case Western Reserve University (Protocol 2013-0016,2013-0017). The animals were subjected to minimal stress during tailvein injections. The 8 week old wild-type C57BL/6 mice (JacksonLaboratory, Bar Harbor, Minn.) were used for all of the in vitro and exvivo tissue staining, and SD rats (Harlan Laboratory, Indianapolis,Ind.) were used for microPET/CT imaging studies. The rats were fastedovernight prior to imaging, but had access to water. Their diet was thenreplenished after microPET/CT imaging.

In Vitro Tissue Staining and Assay of Fluorescent Intensity

Wild-type mice (20-22 g, 8 weeks old) were deeply anesthetized andperfused transcardially with precooled saline (4° C., 10 mL/min for 1min followed by 7 mL/min for 6 min) followed by fixation with precooled4% PFA in PBS (4° C., 10 mL/min for 1 min followed by 7 mL/min for 6min). Brain tissues were then removed, postfixed by immersion in 4% PFAovernight, dehydrated in 10%, 20%, and 30% sucrose solution, embedded ina freezing compound (OCT, Fisher Scientific, Suwanee, Ga.), andsectioned at 20 μm with a cryostat (Thermo HM525, Thermo FisherScientific Inc., Chicago, Ill., USA). Brain sections were collected fromAP (1.0) to AP (−0.1) and in 12 sections were mounted in order on thebottom of 12 superfrost slides (Fisher Scientific) with one section oneach slide. Sections 13-24 were mounted in order on the middle of eachslide, and sections 25-36 were mounted in order on the top of eachslide. Sections were then incubated with tested compounds (1 mM, 5% DMSOin 1×PBS (pH 7.0), 6 sections per compound) for 25 min at roomtemperature in the dark. Excess compounds were washed by briefly rinsingthe slides in PBS (lx) and coverslipped with fluoromount-G mountingmedia (Vector Laboratories, Burlingame, Calif.). Sections were thenexamined under a microscope (Leica DM4000B, Leica Microsystem Inc.,Buffalo Grove, Ill., USA) equipped for fluorescence (DFC7000T), andimages of the stained mouse whole brain sections were acquired with thesame exposure time. ImageJ software was then used to quantify pixelintensity values on 6 sections of each tested compound. A ROI wasselected on the genus of the corpus callosum (gcc, white matter), andthe same size ROI was applied on the midline between gcc and the edge ofthe section (see FIG. 2A), which is considered as gray matter. Imageswere analyzed by two experienced individuals. The FIR of white matter togray matter were then calculated.

Ex Vivo Imaging

Wild-type mice were administered with the newly synthesized compounds(40 mg/kg) via tail vein injection, and 30 min later, the mice wereperfused transcardially with saline followed by 4% PFA in PBS. Braintissues were then removed, postfixed by immersion in 4% PFA overnight,dehydrated in 30% sucrose solution, cryostat sectioned at 100 μm, andmounted on superfrost slides, and images were acquired directly using aLeica fluorescent microscope.

Radiosynthesis

No carrier-added (n.c.a.) [¹⁸F] fluoride was produced by a cyclotron(Eclipse High Production, Siemens) via the nuclear reaction ¹⁸O(p,n)¹⁸F. At the EOB, the activity of aqueous [¹⁸F] fluoride (50-100mCi) was transferred to the GE Tracerlab F×n synthesizer with highhelium pressure. After delivery, the radioactive solution was passedthrough a Sep-Pak light QMA cartridge (Waters, WAT023525, 130 mg, 37-55μm, preconditioned with 5 mL of water followed by 10 mL of air insyringe) and was eluted with K₂CO₃ solution (6 mg, 0.043 mmol, in 0.6 mLwater) followed by K₂₂₂ solution (12 mg, 0.032 mmol, in 1 mLacetonitrile). The solvent was evaporated under a steam of helium at 85°C. for 5 min, and the residue was vacuumed at 55° C. for another 3 minto get the anhydrous K222/[¹⁸F] complex. A solution of the tosylatedprecursors (3-5 mg, 0.0062-0.011 mmol, in 0.8 mL acetonitrile) was addedto the above dried complex, and the mixture was heated at 110° C. for 10min. Ethyl acetate (3 mL) and hexane (2 mL) were added to the reactionvessel, and the mixture was passed through a preconditioned Sep-Paksilica cartridge (Waters, WAT 020520, 690 mg, 55-105 μm, preconditionedwith 5 mL of ether). The solvent was removed under a steam of helium at70° C., and the residue was added to a tin chloride solution (30 mg,0.16 mmol, in 1 mL ethanol and 0.5 mL HCl (1 M). The resulting mixturewas heated at 115° C. for 10-20 min A NaOH solution (0.8 mL, 1 M) andwater (15 mL) were then added, and the resulting mixture was passedthrough a preconditioned Sep-Pak C-18 cartridge (Waters, WAT020515, 360mg, 55-105 μm, preconditioned with 5 mL of ethanol followed by 10 mL ofwater, then dried by 10 mL of air in a syringe). The cartridge waswashed with another 20 mL of water, and the crude products were elutedwith 1 mL acetonitrile which was further purified by semipreparativeHPLC (Phenomenex C-18, 10×250 mm, acetonitrile:H₂O=60:40, flow rate of 5mL/min, tR=6-14 min). The radioactive fraction containing the desiredproducts was collected, diluted with water, loaded onto a Sep-Pak C-18cartridge, and eluted with 1 mL ethanol. After evaporation, the residuewas redissolved in 5% ethanol in saline solution and filtered (0.22 μm)into a sterile injection bottle for animal use. RCP and specificactivity (SA) were determined by analytical HPLC (Phenomenex C-18,4.6×250 mm, acetonitrile:H₂O=65:35, flow rate of 1 ml/min, tR=6-10 min).SA was calculated by area of the UV peak of purified F-18 compound andtitrated with the standard curve of the nonradioactive referencecompound of known concentration.

MicroPET/CT Image Acquisition and Analysis

MicroPET/CT imaging was performed using a Siemens Inveon microPET/CTscanner in the Case Center for Imaging Research. For better anatomiclocalization, CT coregistration was applied. Before microPET imaging, CTscout views were taken to ensure the brain tissues were placed in thecoscan field of view (FOV) where the highest image resolution andsensitivity are achieved. Under anesthesia, radiotracers (1-2 mCi) wereadministered via tail vein injection and immediately followed by adynamic PET acquisition up to 60 min Once microPET acquisition was done,the rat was moved into the CT field and a two-bed CT scan was performed.A two-dimensional ordered subset expectation maximization (OSEM)algorithm was used for image reconstruction using CT for the attenuationcorrection. For quantitative analysis, the resultant PET images wereregistered to the CT images which enabled us to accurately define theROI and quantify the radioactivity concentrations. In this study, thewhole brain of rat was used as ROI, and the radioactivity concentrationswere determined in terms of SUV.

In Situ Autoradiography

Ex vivo Wild-type mice were euthanized at 10 min post i.v. injection of[¹⁸F]32 (3.0 mCi). The brains were rapidly removed, placed in optimalcutting temperature (OCT) embedding medium and frozen at −20° C. Afterreaching equilibrium at this temperature, the brains were coronallycryostat sectioned at 60 μm on a cryostat and mounted on superfrostslides. After drying by air at room temperature, the slides were put ina cassette and exposed to film to obtain images.

Ex Vivo Block

For ex vivo blocking studies, mice were pretreated with CIC, a compoundwhich has proved to bind to myelin with high affinity and specificity(i.v. 160 mg/kg) 3 h before injection of [¹⁸F]32 (3.0 mCi). Mice werethen euthanized, and brains were removed and sectioned. After drying byair, the slides were put in a cassette and exposed to film to obtainimages.

Biostability Off [¹⁸F]32 in Wild-Type Mice

The in vivo biostability of [¹⁸F]32 in plasma was analyzed usingradio-HPLC. Briefly, mice (n=3) were sacrificed at 5, 30, and 60 minpostinjection of [¹⁸F]32 (0.8-1.0 mCi) through tail vein. Blood wascollected into VACUETTE blood collection tubes which were precoated withK3EDTA (containing 4.0 mg of K3EDTA, Greiner Bio One, Germany). Thesamples were centrifuged at 3000 rpm for 5 min at 4° C. to separateplasma. The supernatant plasma samples were mixed with ice-cold methanoland centrifuged again at 10,000 rpm for 3 min to further remove proteinsand other biological matrix. The supernatant was then analyzed byradio-HPLC using acetonitrile/water (60:40, v/v) as mobile phase at aflow rate of 1.0 mL/min. The percentage of parent compound was thencalculated.

Results Chemistry

The design of the fluorinated radioligands is based on the structure ofMeDAS, a lead myelin-specific radioligand that we previously developedfor PET imaging of myelination. Structure-activity relationship studiessuggested that fluorine could be introduced through alkylation of theamino groups, which are responsible for binding to myelin. Once variousfluorinated alkyl groups are introduced to MeDAS, the derivativesproduced may have different lipophilicity and permeability across theblood-brain barrier (BBB). Thus, the newly synthesized fluorinatedanalogs, even though they share the same pharmacophore as MeDAS, maydisplay distinctly different physicochemical and biological properties.Thus, we conducted a systematic evaluation of the in vitro and in vivoproperties of binding to myelin.

Our previous structure-activity relationship studies suggested that thetwo amino groups can be modified but cannot be replaced, as they bothare responsible for myelin binding. Thus, we introduced fluorine toMeDAS via an aliphatic ether side chain in the beta-position to theamino group. Introduction of fluorine often reduces the lipophilicity ofcompounds. We thus synthesized a series of fluorinated MeDAS analogs byalkylating the other amino group opposite to the fluorinated alkyl aminogroup.

The lipophilicity of these compounds was calculated using ALOGPS 2.1program (Virtual Computational Chemistry Laboratory). As shown in Table1, the calculated lipophilicity (c Log P) of these newly synthesizedcompounds ranges from 2.7 to 5.4. Such a range provides a good spectrumof lipophilicity for us to navigate the in vitro and in vivo bindingproperties.

TABLE 1 Entry Structure clogP MeDAS

3.39 25

2.78 32

3.65 33

4.23 34

4.68 35

4.48 36

5.27 37

2.75 39

3.65

As shown in Scheme 1, various tosylated nitrobenzaldehydes (3, 4, 6) andfluorinated nitrobenzaldehydes (5, 7) were first prepared from3-hydroxy-4-nitrobenzaldehyde (1) and 4-hydroxy-3-nitrobenzaldehdye (2)in 50-90% yield.

For the synthesis of fluorinated MeDAS analogs except compound 25, westarted with p-aminobenzyldiethyl phosphonate. As shown in Scheme 2, theamino group was first protected with Boc to generate compound 9 in 95%yield, which was subsequently alkylated with different alkyl iodides toobtain compounds 10-14 in 20 to 80% yield.

The Boc-protected-N-alkylated diethyl benzylphosphonate and diethyl(4-nitrobenzyl)phosphonate (10-15) were coupled with tosylatednitrobenzaldehydes (3, 4, 6) to produce the radiolabeling precursors(16-23) through the Homer-Wadsworth-Emmons reaction (Schemes 3 and 4) in40 to 80% yield. These precursors (16-23) were used for theradiosynthesis of F-18-labeled MeDAS analogs.

For the synthesis of compound 25, the tosylated compound 23 wassubjected to a nucleophilic substitution to generate compound 24 in 80%yield followed by reduction of the nitro group to give the final coldstandard compound 25 in 50% yield (Scheme 4).

For the synthesis of the remainder of fluorinated compounds 32-37 and39, fluorinated nitrobenzaldehydes (5, 7) and compounds 10-15 werecoupled to produce intermediates 26-31 and 38 in 40-60% yield (Scheme5). This was followed by a one-pot reaction using SnCl₂ in ethanol andethyl acetate, which allowed for simultaneous deprotection of the Bocgroup and reduction of the nitro group to the amino group of compounds26-31 and 38 in 30-90% yield to generate final compounds 32-37 and 39that can be used as standards for radiochemistry synthesis as well asfor biological evaluations.

Similar to MeDAS, all of the newly synthesized fluorinated analogs arefluorescent. The excitation/emission spectra were acquired inacetonitrile for comparison as shown in FIG. 1. The excitationwavelengths of these compounds were measured to be 390±10 nm, and theemission wavelengths were measured to be 420±10 nm. Such wavelengths arein an ideal range to conduct fluorescent tissue staining which allowsexamination of the preliminary binding specificity for myelin, either invitro or in situ.

In vitro Staining and Assay of the Fluorescent Intensity. In vitrofluorescent tissue staining provides a convenient way to screen bindingspecificity of the newly synthesized compounds for myelin. Myelinsheaths are distributed more dominantly in the white matter than in thegray matter. Thus, the fluorescent intensity is expected to beconsistent with the pattern of myelin distribution in the brain. Asexpected, in vitro tissue staining of mouse brain sections showed thatall target compounds (25, 32-37, and 39) selectively stained myelinatedregions such as corpus callosum and striatum. In order to compare thebinding feature for myelin by in vitro histological staining of mousebrain tissue sections, all compounds were tested at same time, theconcentration of all the tested staining solution was 1 mM, and sectionswere imaged under the same exposure time after being incubated with thestaining solution for 25 min. Next, we selected a representative regionin the genus of the corpus callosum (gcc) and a representative region inthe subcortical gray matter (cortex) and calculated the fluorescentintensity ratio (FIR) between both regions (FIG. 2A). This allowed us topreliminarily compare the binding specificity for myelin. As shown inFIG. 2B, the newly synthesized compounds can be divided into two groups,with two of the compounds (32 and 35) showing a FIR>2 and the rest ofthem <2. The higher FIR indicates a higher degree of specific binding.This study suggested that compounds 32 and 35 be the lead candidates forfurther evaluation.

Ex Vivo Imaging

The newly synthesized fluorescent compounds are also suitable for insitu tissue staining through direct tail vein injection. This studyallowed us to determine both brain permeability as well as in vivobinding specificity. Thus, ex vivo imaging was performed following invitro tissue staining. At a dose of 40 mg/kg, each tested compound wasadministered to mice through tail vein injection. Use of highconcentration is needed to enable fluorescence visualization ex vivo. Asshown in FIG. 3, the two lead compounds readily enter the brain andspecifically bind to myelin tracts present in the white matter regionssuch as the corpus callosum and striatum. In fact, all the newlysynthesized compounds can penetrate the BBB and selectively localize inthe white matter region. Such in situ staining, however, is only aqualitative and insensitive measure to determine brain permeability. Theinjected amounts are in the range of 40 mg/kg, which is at least 3orders of magnitude greater than in vivo PET imaging.

Radiosynthesis

Encouraged by the above results, we then evaluated the in vivopharmacokinetic profiles of the newly synthesized compounds labeled withpositron emitting fluorine-18. The radiosynthesis was achieved throughnucleophilic substitution of a tosylate group, as shown in Schemes 6, 7,and 8, with fluorine-18 generated by an onsite cyclotron followed by areduction and/or acidic hydrolysis of the Boc protection group. In thisstudy, the tosylated precursor was employed in a three-stepradiosynthesis starting with a nucleophilic substitution with ¹⁸F⁻ inthe presence of K₂CO₃ and Kryptofix (K₂₂₂) in MeCN at 115° C. for 10min. After evaporation of MeCN, more than 91±7% (n=15) of the activitywas retained in the reaction vessel. The fluorinated intermediate wassimply purified by passing through a silica Sep-Pak to remove unreactedfree fluorine-18. The fluorinated intermediate (60-80%) was then reducedby SnCl₂ in hydrochloric acid and ethanol at 120° C. for 10 min to yieldthe primary amine (compound 25 and 37). At this step, it was necessaryto extend the reaction time if the acidic hydrolysis of the Bocprotecting group is needed (compound 32-36 and 39). After cooling toroom temperature, the reactant was neutralized to pH 8-9 by addition ofNaOH (1.0 M, 0.8 mL) and was further purified by semipreparative HPLC toyield the final products with modest yields (for the three-stepprocedure) ranging from 30-60% (decay corrected to the end ofbombardment (EOB)) within 120-130 min.

The identities of the products were confirmed using HPLC by co-injectionwith each cold standard compound. Radio-chemical purity (RCP) of finalproducts was over 98% determined by analytical radio-HPLC. The specificactivity at the end of synthesis was in a range of 0.55-2.5 Ci/μmol. Allthe [¹⁸F]⁻ labeled compounds were stable after being allowed to stand atroom temperature for 4 h or diluted with saline. The radiochemicalpurity of both the original and diluted aqueous solutions was >95%determined by analytical HPLC.

Quantitative microPET/CT Imaging

Following radio-labeling, the brain entry, retention, and clearance ofeach compound was determined by microPET/CT imaging in Sprague-Dawley(SD) rats. For quantitative analysis, the resultant microPET images wereregistered to the CT images, which allowed us to accurately define theregion of interest (ROI) and quantify the radioactivity concentrationsof each compound. The radioactivity concentrations were determined interms of standardized uptake values (SUV). As shown in FIG. 4A, all ofthe compounds entered the brain at early time points with variousretention and clearance rates at later time points. Similar to[¹¹C]MeDAS, the radioactivity concentration rapidly reached a peakwithin 5 min and then decreased to reach a plateau at 40-60 min. Most ofthe F-18-labeled compounds exhibited a relatively low retention and fastclearance, which suggested either slow interaction with myelin membraneor low binding potency. The two lead candidates (compounds 32 and 35)that were identified through fluorescent tissue staining showed distinctin vivo pharmaco-kinetics. [¹⁸F]32 showed the highest brain uptake atearly time points and highest retention at later time points with aclearance rate of 2.62, which suggests a low nonspecific binding. Incomparison [¹⁸F]35 showed relatively high retention at later timepoints, but the brain uptake was significantly lower than [¹⁸F]32 with aclearance rate of 0.70, which suggests a relatively high nonspecificbinding. As shown in FIG. 4B, although [¹⁸F]36 displayed the secondhighest retention at later time points, the initial brain uptake was thelowest compared with the rest of the compounds with a clearance rate of0.30, which suggests poor brain permeability or high nonspecificbinding. In addition, due to the highest c Log P value (5.27), [¹⁸F]36displayed very slow washout from the whole brain, indicating it ishardly cleared from the brain once penetrating into the BBB. As shown inFIG. 4A, both [¹¹C]MeDAS and [¹⁸F]32 showed similar brain entry at earlytime points. Yet, the clearance of [¹⁸F]32 was found to be faster thanthat of [¹¹C]MeDAS. Overall, among the radiolabeled analogs, [¹⁸F]32clearly stands out as the best lead candidate for in vivo imaging ofmyelin, which is consistent with in vitro FIR data. RepresentativemicroPET/CT images of [¹⁸F]32 are shown in FIG. 5.

In Situ Autoradiography

To validate the PET results and confirm that the PET signals were indeedfrom specific binding to myelin, we conducted ex vivo autoradiography.Auto-radiography allowed us to examine microscopic localization of theradiolabeled compounds after brain entry. After quantitative analysis ofmicroPET/CT studies, [¹⁸F]32 was selected to perform ex vivo filmautoradiography, which allowed us to further examine brain permeabilityand specific binding of the compound. Thus, we conducted ex vivoautoradiography studies in the mouse brain by administering [¹⁸F]32through tail vein injections. As shown in FIG. 6A, [¹⁸F]32 is localizedpredominantly in the white matter region which is consistent with thepattern of myelin distribution. A relatively distinct labeling of thecorpus callosum, an area known to have a high density of myelinatedsheaths, was observed after mouse brain tissue sections (coronal) wereexposed to film for 10 min. The autoradiographic visualization wasconsistent with histological staining of myelinated regions (FIG. 6C).To demonstrate that radioactivity in the autoradiography was fromspecific binding to myelin, we pretreated rats with nonlabeled CIC, amyelin-specific agent that we previously developed. As shown in FIG. 6B,pretreatment of CIC significantly reduces the contrast of radioactivityin gcc vs cortex. Since CIC itself is also fluorescent, a distinctstaining of CIC can be examined on the same section when checked under afluorescence microscope (FIG. 6D). When the film was analyzed usingImageJ, the optical density ratio (ODR) of gcc to cortex was employed todetermine the radiographic staining ratio between white matter and graymatter. Statistical analysis of ODR on the film showed there is asignificant difference (p<0.05) between control (3.73±0.31) and ex vivoblock studies (1.91±0.04) (FIG. 6E). Such an ex vivo competition studysuggests that [¹⁸F]32 readily enters the brain and specifically binds tomyelin sheaths.

Biostability of [¹⁸F]32

Because [¹⁸F]32 showed the highest brain uptake and fastest washout innormal mice, we further evaluated the in vivo biostability of [¹⁸F]32 inplasma. After injection of [¹⁸F]32, the plasma samples were harvestedand analyzed by radio-HPLC. To the plasma samples were first addedice-cold methanol to precipitate proteins and other biohydrophilicmatrix components. The mixtures were centrifuged at 10,000 rpm for 5min. The supernanent were then separated and loaded onto radio-HPLC forassessment (Phenomenex C-18, 4.6×250 mm, acetonitrile:H₂O=65:35, flowrate of 1 mL/min). Similar to [¹¹C]PIB and [¹⁸F]-Flutemetamol, [¹⁸Fafter tail vein injection. The percentage of parent [¹⁸F]32 in plasmawas determined to be 78.14±9.15% at 5 min after injection. Thepercentage of parent [¹⁸F]32 decreased to 53.12±7.31% and 32.45±5.80% at30 and 60 min postinjection. All the metabolites found in the plasmawere hydrophilic with retention time close to void volume, which areincapable of penetrating the BBB.

Example 2

We designed and synthesized a novel series of fluorinated andfluorescent compounds using click chemistry through Cu(I)-catalyzedHuisgen cycloaddition. In this example, we show the design, synthesis,and imaging studies of a series of fluorinated triazole analogues offluorescent trans-stilbene. We show that, using the same imaging agent,PET can be coregistered with microscopic 3D cryoimaging to seamlesslycombine the unique features of quantitative physiologic informationprovided by PET with microscopic histologic information in highresolution provided by cryoimaging.

Methods and Materials

Chemicals and reagents were used as received without furtherpurification. Glassware was dried in an oven at 130° C. and purged witha dry atmosphere prior to use. Unless otherwise mentioned, reactionswere performed open to air. Reactions were monitored by TLC andvisualized by a dual short/long wave UV lamp. Column flashchromatography was performed using 230-400 mesh silica gel (Fisher).Preparative TLC was performed on Analtech Preparative TLC Uniplates withUV254 fluorescence indicator (500 μm). NMR spectra were recorded on aVarian Inova 400 spectrometer and 500 MHz Bruker Ascend Avance III HD atroom temperature. Florescence spectra were recorded, and chemical shiftsfor ¹H and ¹³C NMR were reported as 6, part per million (ppm), andreferenced to an internal deuterated solvent central line. Theabbreviations s, br s, d, dd, ddd, br d, t, dt, q, m, and br m stand fortheir resonance multiplicity singlet, broad singlet, doublet, doublet ofdoublets, doublet of doublet of doublets, broad doublets, triplet,triplets of doublets, quartet, multiplet, and broad multiplet,respectively, which were calculated automatically on a MestReNova 10.0.The purity of tested compounds as determined by analytical HPLC was 95%.HRMS-ESI mass spectra were acquired on an Agilent Q-TOF. UV absorptionwas measured on a Cary 50 Bio spectrophotometer using a standard 1 cm×1cm quartz cuvette. Fluorescence was measured with a Cary Eclipsespectrophotometer using a 1 cm×1 cm quartz cuvette.

General Method for Alkylation of tert-Butyl(4-((Diethoxyphosphoryl)methyl)phenyl)carbamate (3-5)

To an oven-dried 100 mL round-bottom flask purged with argon gas andfitted with a magnetic stir bar, sodium hydride (2 equiv 95%) andtert-butyl (4-((diethoxyphosphoryl) methyl)phenyl)carbamate (1 equiv)were added together with dry THF (25 mL) at 0° C. and purged with argongas. Iodo-alkane (3 equiv) was added slowly dropwise after 30 min at 0°C. under argon gas. The reaction was stirred under argon and allowed toreach room temperature overnight. After completion, the reaction wasquenched with water and THF was removed in vacuum. The residue wasdissolved in DCM and water and the aqueous layer was extracted threetimes with DCM (30 mL). The organic layers were combined and washedtwice with water (50 mL) and once with brine (50 mL). The organic layerwas dried over Na₂SO₄ and concentrated to give the desired product as asticky oil, which was used for the next step without furtherpurification.

General Method for Horner-Wadsworth-Emmons Coupling Reaction (6-11)

To an oven-dried 100 mL round-bottom flask with a magnetic stir bar wasadded sodium hydride (2 equiv, 95%). The flask was purged with argon,and 2 mL of dry DMF was added. This solution was cooled to 0° C., andthe phosphonate in DMF (2 mL) was added. The mixture was allowed to stirfor 1 h at 0° C. Then the aldehyde in 2 mL of DMF was added slowly. Thereaction was continued for another 2 h at 0° C. under argon followed byquenching with water (50 mL) and then extracted with ethyl acetate (50mL) three times. The organic layer was washed with saturated NaHCO₃ (20mL) and brine (50 mL), dried with Na₂SO₄, and concentrated to give thecrude product, which was purified with flash chromatography usinghexanes and ethyl acetate as eluents.

tert-Butyl (E)-Ethyl(4-(4-(prop-2-yn-1-yloxy)styryl)phenyl)-carbamate(7)

¹H NMR (400 MHz, chloroform-d) δ 7.43-7.36 (m, 4H), 7.13 (d, J=8.2 Hz,2H), 6.98 (d, J=16.3 Hz, 1H), 6.93 (d, J=1.9 Hz, 2H), 6.90 (d, J=7.4 Hz,1H), 4.63 (dd, J=2.5, 1.1 Hz, 2H), 3.65 (q, J=7.1 Hz, 2H), 2.50 (t,J=2.4 Hz, 1H), 1.42 (s, 9H), 1.13 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz,chloroform-d) δ 157.4, 154.6, 141.7, 135.3, 131.1, 128.1, 127.9, 127.2,126.7, 126.5, 115.3, 80.2, 78.7, 76.0, 56.0, 45.1, 28.6, 14.2.

tert-Butyl (E)-Propyl(4-(4-(prop-2-yn-1-yloxy)styryl)phenyl)-carbamate(8)

¹H NMR (400 MHz, chloroform-d) δ 7.40 (dd, J=8.7, 2.1 Hz, 4H), 7.14 (d,J=8.1 Hz, 2H), 6.99 (d, J=16.5 Hz, 1H), 6.93 (s, 2H), 6.90 (d, J=7.1 Hz,1H), 4.63 (d, J=2.4 Hz, 2H), 3.62-3.54 (m, 2H), 2.50 (s, 1H), 1.61-1.49(m, 2H), 1.43 (s, 9H), 0.87 (t, J=7.4 Hz, 3H). ¹³C NMR (100 MHz,chloroform-d) δ 157.4, 154.9, 141.8, 135.3, 131.1, 128.2, 127.9, 127.3,126.7, 126.5, 115.3, 80.2, 78.7, 76.0, 55.99, 51.8, 28.6, 22.0, 11.4.

tert-Butyl(E)-(4-(4-((tert-Butoxycarbonyl)(prop-2-yn-1-yl)amino)-styryl)phenyl)(methyl)carbamate(9)

¹H NMR (400 MHz, chloroform-d) δ 7.46 (dd, J=8.7, 6.7 Hz, 4H), 7.31 (d,J=8.2 Hz, 2H), 7.22 (d, J=8.6 Hz, 2H), 7.03 (s, 2H), 4.37 (d, J=2.5 Hz,2H), 3.26 (s, 3H), 2.27-2.24 (t, J=4.8 1H), 1.46 (s, 18H). ¹³C NMR (100MHz, chloroform-d) δ 154.8, 154.1, 143.3, 141.5, 135.4, 134.4, 128.2,127.9, 127.8, 126.9, 126.8, 126.5, 125.6, 81.4, 80.6, 80.2, 72.1, 39.9,37.4, 28.5, 28.5.

tert-Butyl(E)-(4-(4-((tert-Butoxycarbonyl)(prop-2-yn-1-yl)amino)-styryl)phenyl)(ethyl)carbamate(10)

¹H NMR (400 MHz, chloroform-d) δ 7.45-7.42 (m, 4H), 7.28 (d, J=8.4 Hz,2H), 7.14 (d, J=8.5 Hz, 2H), 7.01 (s, 2H), 4.32 (d, J=2.4 Hz, 2H), 3.65(q, J=7.0 Hz, 2H), 2.23 (t, J=2.4 Hz, 1H), 1.44 (s, 9H), 1.44 (s, 9H),1.12 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, chloroform-d) δ 154.6, 154.1,142.0, 141.6, 135.4, 135.0, 128.3, 128.0, 127.2, 126.9, 126.9, 126.5,81.4, 80.3, 80.2, 72.1, 45.0, 39.9, 28.6, 28.5, 14.1.

tert-Butyl(E)-(4-(4-((tert-Butoxycarbonyl)(prop-2-yn-1-yl)amino)-styryl)phenyl)(propyl)carbamate(11)

¹H NMR (400 MHz, chloroform-d) δ 7.48-7.45 (m, 4H), 7.31 (d, J=8.4 Hz,2H), 7.18 (d, J=8.3 Hz, 2H), 7.05 (s, 2H), 4.37 (d, J=2.5 Hz, 2H),3.62-3.58 (m, 2H), 2.26 (t, J=2.4 Hz, 1H), 1.63-1.52 (m, 2H), 1.47 (s,9H), 1.44 (s, 9H), 0.89 (t, J=7.4 Hz, 3H). 13C NMR (100 MHz,chloroform-d) δ 154.9, 154.1, 142.2, 141.6, 135.4, 135.0, 128.3, 128.0,127.3, 126.9, 126.5, 81.4, 80.2, 80.2, 72.0, 51.7, 39.9, 28.5, 28.4,21.9, 11.4.

General Method for Click Chemistry (12-17)

A portion of 1-(4-methylbenzenesulfonate)-2-uoroethanol (1.67 equiv) inDMF (4 mL) was stirred with a suspension of sodium azide (5.6 equiv) atroom temperature. After 48 h, the solution was filtered through Celiteand the crude 1-azido-2-fluoroethane was used immediately in the nextstep without further purification. In a separate flask, copper(I) iodide(5.4 equiv) was suspended in methanol (2 mL) under an argon atmospherewith vigorous stirring. In rapid succession, the alkyne precursor (1equiv) dissolved in methanol (1 mL), the crude 1-azido-2-fluoroethanedissolved in DMF, and triethylamine (5.4 equiv) were added. The reactionmixture was stirred overnight at room temperature, and saturated NaHCO₃(20 mL) was then added followed by extraction with ethyl acetate (20 mL)three times. The combined organic layers were washed with water (50 mL)and brine (50 mL), dried with Na₂SO₄, and concentrated to give the crudeproduct, which was purified by column chromatography eluted with hexanesand ethyl acetate.

tert-Butyl(E)-(4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)-methoxy)styryl)phenyl)(methyl)carbamate(12)

¹H NMR (400 MHz, chloroform-d) δ 7.70 (d, J=1.0 Hz, 1H), 7.40 (dd,J=8.7, 3.0 Hz, 4H), 7.17 (d, J=8.5 Hz, 2H), 6.95 (s, 3H), 6.93 (d,J=22.8 Hz, 4H), 6.90 (d, J=16.5 Hz, 2H), 4.81 (dd, J=5.2, 4.1 Hz, 1H),4.67 (ddd, J=14.2, 5.5, 4.3 Hz, 2H), 4.59 (dd, J=5.2, 3.9 Hz, 1H), 3.22(s, 3H), 1.42 (s, 9H). ¹³C NMR (100 MHz, chloroform-d) δ 158.0, 154.9,144.6, 143.0, 134.7, 130.8, 128.0, 127.9, 126.5, 126.4, 125.7, 124.0,115.2, 114.8, 82.5, 80.8, 80.6, 62.1, 50.9, 50.7, 37.4, 28.5.

tert-Butyl(E)-(4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4yl)-methoxy)styryl)phenyl)(ethyl)carbamate(13)

¹H NMR (400 MHz, chloroform-d) δ 7.72-7.70 (m, 1H), 7.40 (dd, J=8.6, 1.8Hz, 4H), 7.12 (d, J=8.1 Hz, 2H), 6.98 (d, J=16.3 Hz, 1H), 6.96-6.93 (m,2H), 6.91 (d, J=15.9 Hz, 1H), 5.19 (s, 2H), 4.81 (dd, J=5.2, 4.1 Hz,1H), 4.75-4.65 (m, 2H), 4.59 (dd, J=5.2, 4.0 Hz, 1H), 3.64 (q, J=7.1 Hz,2H), 1.40 (s, 9H), 1.12 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz,chloroform-d) δ 158.0, 154.7, 144.6, 141.7, 135.3, 130.8, 128.1, 127.9,127.2, 126.7, 126.4, 124.0, 115.2, 114.8, 82.5, 80.8, 80.2, 62.1, 50.9,50.7, 45.1, 28.6, 14.1.

tert-Butyl(E)-(4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)-methoxy)styryl)phenyl)(propyl)carbamate(14)

¹H NMR (400 MHz, chloroform-d) δ 7.70 (s, 1H), 7.40 (dd, J=8.7, 1.9 Hz,4H), 7.12 (d, J=8.1 Hz, 2H), 6.98 (d, J=16.2 Hz, 1H), 6.96-6.93 (m, 2H),6.91 (d, J=16.2 Hz, 1H), 5.18 (s, 2H), 4.80 (dd, J=5.2, 4.1 Hz, 1H),4.67 (ddd, J=14.2, 5.4, 4.3 Hz, 2H), 4.58 (dd, J=5.2, 4.0 Hz, 1H),3.59-3.52 (m, 2H), 1.57-1.47 (m, 2H), 1.40 (s, 9H), 0.84 (t, J=7.4 Hz,3H). ¹³C NMR (100 MHz, chloroform-d) δ 158.1, 154.9, 144.7, 141.8,135.3, 130.9, 128.1, 127.9, 127.3, 126.7, 126.4, 124.0, 115.2, 114.8,82.5, 80.8, 80.2, 62.2, 51.7, 50.9, 50.7, 28.5, 21.9, 11.4.

tert-Butyl(E)-(4-(4-((tert-Butoxycarbonyl)(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-styryl)phenyl)(methyl)carbamate(15)

¹H NMR (400 MHz, chloroform-d) δ 7.68 (s, 1H), 7.44 (dd, J=8.7, 6.9 Hz,4H), 7.28 (d, J=7.8 Hz, 2H), 7.22 (d, J=8.6 Hz, 2H), 7.02 (s, 2H), 4.92(s, 2H), 4.84 (dd, J=5.2, 4.1 Hz, 1H), 4.71 (ddd, J=13.2, 5.3, 4.3 Hz,2H), 4.62 (dd, J=5.1, 4.1 Hz, 1H), 3.27 (s, 3H), 1.46 (s, 9H), 1.45 (s,9H). ¹³C NMR (100 MHz, chloroform-d) δ 154.6, 154.5, 145.9, 143.3,142.1, 135.0, 134.5, 128.1, 127.9, 126.8, 126.7, 126.4, 125.6, 123.9,82.6, 81.2, 80.9, 80.6, 50.8, 50.6, 46.0, 37.4, 28.5, 28.5.

tert-Butyl(E)-(4-(4-((tert-Butoxycarbonyl)((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-styryl)phenyl)(ethyl)carbamate(16)

¹H NMR (400 MHz, chloroform-d) δ 7.73-7.62 (m, 1H), 7.45 (t, J=8.5 Hz,4H), 7.31-7.26 (m, 2H), 7.17 (d, J=8.5 Hz, 2H), 7.03 (s, 2H), 4.92 (s,2H), 4.82 (dd, J=5.2, 4.1 Hz, 1H), 4.69 (ddd, J=12.4, 5.5, 4.3 Hz, 2H),4.61 (dd, J=5.2, 4.0 Hz, 1H), 3.68 (q, J=7.1 Hz, 2H), 1.45 (s, 9H), 1.44(s, 9H), 1.16 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, chloroform-d) δ154.6, 154.45, 145.9, 142.1, 141.95, 135.0, 128.1, 128.0, 127.2, 126.9,126.8, 126.4, 123.9, 82.6, 81.2, 80.9, 80.3, 50.8, 50.6, 46.0, 45.0,28.5, 28.5, 14.1.

tert-Butyl(E)-(4-(4-((tert-Butoxycarbonyl)((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)-styryl)phenyl)(propyl)carbamate(17)

¹H NMR (400 MHz, chloroform-d) δ 7.68 (s, 1H), 7.45 (dd, J=9.0, 7.2 Hz,4H), 7.28 (d, J=9.0 Hz, 2H), 7.17 (d, J=8.4 Hz, 2H), 7.03 (s, 2H), 4.92(s, 2H), 4.84 (dd, J=5.2, 4.1 Hz, 1H), 4.71 (ddd, J=13.2, 5.5, 4.3 Hz,2H), 4.63 (dd, J=5.2, 4.0 Hz, 1H), 1.62-1.53 (m, 2H), 1.45 (s, 9H), 1.44(s, 9H), 0.89 (t, J=7.4 Hz, 3H), 0.89 (t, J=12.0 Hz, 3H). ¹³C NMR (100MHz, CDCl₃) δ 154.9, 154.5, 146.0, 142.1, 135.0, 128.1, 128.0, 127.3,126.9, 126.4, 123.9, 82.6, 81.2, 80.9, 80.26, 51.7, 50.8, 50.6, 46.0,28.5, 28.5, 21.9, 11.4.

General Method for Boc Deprotection (18-23)

The click product was dissolved in 2 mL of methanol. To this mixture wasadded 2 mL of HCl (1.2 M), and the mixture was stirred for 1.5 h at 60°C. and then sufficient NaOH (1 M) was added to bring the pH to 10. Waterwas added to the mixture and extracted with ethyl acetate (10 mL) threetimes, washed with brine (30 mL), dried with Na₂SO₄, and concentrated togive the crude product. Prep TLC or flash chromatography(hexanes/acetone) yielded the deprotected product.

(E)-4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)styryl)-N-methylaniline(18)

¹H NMR (400 MHz, DMSO-d₆) δ 8.27 (s, 1H), 7.44 (d, J=8.8 Hz, 2H), 7.31(d, J=8.6 Hz, 2H), 7.07-6.98 (m, 2H), 6.94 (d, J=16.4 Hz, 1H), 6.85 (d,J=16.4 Hz, 1H), 6.52 (d, J=8.7 Hz, 2H), 5.82 (q, J=5.0 Hz, 1H), 5.16 (s,2H), 4.89 (dd, J=5.3, 4.1 Hz, 1H), 4.81-4.75 (m, 2H), 4.73-4.67 (m, 1H),2.69 (d, J=5.1 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 157.4, 149.9,143.4, 131.5, 127.8, 127.5, 127.4, 125.4, 125.3, 122.9, 115.3, 112.1,83.0, 81.7, 61.5, 50.6, 50.4, 30.1. HR-MS (ESI) m/z calculated for(C₂₀H₂₂FN₄O) [M+H]⁺ 353.1772, found 353.1773. HPLC purity: 96.94%,retention time 4.00 min C-18 reversed-phase HPLC (Phenomenex, 10 mm×250mm); eluent, acetonitrile:H₂O=40:60; flow rate of 1.0 mL/min.

(E)-4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)styryl)-N-ethylaniline(19)

¹H NMR (500 MHz, chloroform-d) δ 7.74 (s, 1H), 7.40 (d, J=8.1 Hz, 2H),7.33 (d, J=8.0 Hz, 2H), 6.96 (d, J=8.1 Hz, 2H), 6.90 (d, J=16.3 Hz, 1H),6.84 (d, J=16.3 Hz, 1H), 6.59 (d, J=8.0 Hz, 2H), 5.24 (s, 2H), 4.85 (t,J=4.8 Hz, 1H), 4.76 (t, J=4.8 Hz, 1H), 4.71 (t, J=4.8 Hz, 1H), 4.65 (t,J=4.8 Hz, 1H), 3.18 (q, J=7.1 Hz, 2H), 1.27 (t, J=6.7 Hz, 3H). ¹³C NMR(125 MHz, chloroform-d) δ 157.2, 147.8, 144.7, 131.6, 127.5, 127.3,127.2, 126.9, 123.8, 123.7, 115.0, 112.8, 82.2, 80.8, 62.0, 50.7, 50.5,38.5, 14.8. HR-MS (ESI) m/z calculated for (C₂₁H₂₄FN₄O) [M+H]⁺ 367.1929,found 367.1932. HPLC purity: 96.22%, retention time 4.02 min C-18reversed-phase HPLC (Phenomenex, 10 mm×250 mm); eluent,acetonitrile:H₂O=40:60; flow rate of 1.0 mL/min.

(E)-4-(4-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)styryl)-N-propylaniline(20)

¹H NMR (400 MHz, chloroform-d) δ 7.68 (s, 1H), 7.33 (d, J=8.7 Hz, 2H),7.25 (d, J=8.5 Hz, 2H), 6.89 (d, J=8.7 Hz, 2H), 6.83 (d, J=16.2 Hz, 1H),6.77 (d, J=16.3 Hz, 1H), 6.51 (d, J=8.5 Hz, 2H), 5.17 (s, 2H), 4.80 (dd,J=5.2, 4.1 Hz, 1H), 4.67 (dt, J=13.4, 4.6 Hz, 2H), 4.61-4.56 (m, 1H),3.04 (t, J=7.1 Hz, 2H), 1.57 (p, J=7.3 Hz, 2H), 0.93 (t, J=7.4 Hz, 3H).¹³C NMR (100 MHz, chloroform-d) δ 157.4, 148.2, 144.9, 131.8, 127.7,127.4, 127.3, 126.9, 123.9, 115.1, 112.9, 82.5, 80.8, 62.2, 50.9, 50.7,45.9, 22.9, 11.8. HR-MS (ESI) m/z calculated for (C₂₂H₂₆FN₄O) [M+H]⁺381.2085, found 381.2090. HPLC purity: 96.28%, retention time 6.50 min.C-18 reversed-phase HPLC (Phenomenex, 10 mm×250 mm); eluent,acetonitrile:H₂O=40:60; flow rate of 1.0 mL/min.

(E)-N-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(4-(methylamino)styryl)aniline(21)

¹H NMR (400 MHz, chloroform-d) δ 7.55 (s, 1H), 7.32 (dd, J=8.6, 1.8 Hz,4H), 6.83 (d, J=1.7 Hz, 2H), 6.64 (d, J=8.6 Hz, 2H), 6.58 (d, J=8.6 Hz,2H), 4.82 (dd, J=5.1, 4.2 Hz, 1H), 4.71 (dd, J=5.2, 4.1 Hz, 1H), 4.65(dd, J=5.2, 4.2 Hz, 1H), 4.59 (dd, J=5.2, 4.1 Hz, 1H), 2.85 (s, 3H). 13CNMR (100 MHz, chloroform-d) δ 148.7, 146.7, 146.7, 128.6, 127.6, 127.4,127.4, 125.5, 124.7, 122.7, 113.5, 112.7, 82.6, 80.8, 50.8, 50.6, 40.1,30.9. HR-MS (ESI) m/z calculated for (C₂₀H₂₃FN₅) [M+H]⁺ 352.1932, found352.1933. HPLC purity: 95.64%, retention time 4.47 min. C-18reversed-phase HPLC (Phenomenex, 10 mm×250 mm); eluent,acetonitrile:H₂O=50:50; flow rate of 1.0 mL/min.

(E)-N-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(4-(ethylamino)styryl)aniline(22)

¹H NMR (400 MHz, chloroform-d) δ 7.57 (s, 1H), 7.31 (dd, J=8.5, 3.6 Hz,4H), 6.82 (d, J=1.5 Hz, 2H), 6.64 (d, J=8.5 Hz, 2H), 6.58 (d, J=8.5 Hz,2H), 4.89-4.82 (m, 1H), 4.72 (dd, J=5.2, 4.0 Hz, 1H), 4.69-4.63 (m, 1H),4.63-4.56 (m, 1H), 4.49 (s, 2H), 3.18 (q, J=7.1 Hz, 2H), 1.26 (t, J=7.1Hz, 3H). ¹³C NMR (100 MHz, chloroform-d) δ 147.8, 146.8, 146.65, 128.6,127.5, 127.4, 127.4, 125.5, 124.6, 122.7, 113.5, 113.0, 82.6, 80.7,50.9, 50.6, 40.1, 38.7, 15.1. HR-MS (ESI) m/z calculated for (C₂₁H₂₅FN₅)[M+H]⁺ 366.2089, found 366.2089. HPLC purity: 98.41%, retention time5.05 min C-18 reversed-phase HPLC (Phenomenex, 10 mm×250 mm); eluent,acetonitrile:H2O=30:70; flow rate of 1.0 ml/min.

(E)-N-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(4-(propylamino)styryl)aniline(23)

¹H NMR (400 MHz, chloroform-d) δ 7.57 (s, 1H), 7.31 (dd, J=8.5, 5.1 Hz,4H), 6.82 (d, J=1.8 Hz, 2H), 6.65 (d, J=8.4 Hz, 2H), 6.58 (d, J=8.5 Hz,2H), 4.84 (t, J=4.6 Hz, 1H), 4.76-4.70 (m, 1H), 4.70-4.64 (m, 1H),4.64-4.58 (m, 1H), 4.49 (s, 2H), 3.10 (t, J=7.1 Hz, 2H), 1.65 (q, J=7.2Hz, 3H), 1.00 (t, J=7.4 Hz, 3H). ¹³C NMR (125 MHz, chloroform-d) δ147.6, 146.6, 146.5, 128.5, 127.3, 127.2, 127.2, 125.4, 124.4, 122.5,113.3, 112.8, 82.2, 80.8, 50.6, 50.5, 45.8, 39.9, 31.9, 22.7, 11.6.HR-MS (ESI) m/z calculated for (C₂₂H₂₇FN₅) [M+H]⁺ 380.2245, found380.2248. HPLC purity: 99.93%, retention time 8.13 min. C-18reversed-phase HPLC (Phenomenex, 10 mm×250 mm); eluent,acetonitrile:H₂O=30:70; flow rate of 1.0 mL/min.

tert-Butyl (4-Formylphenyl)(prop-2-yn-1-yl)carbamate (24)

To an oven-dried 100 mL round-bottom flask purged with argon gas andfitted with a magnetic stir bar was added sodium hydride (140 mg, 95%)and tert-butyl (4-formylphenyl)carbamate (1.02 g), which was purged withargon gas under dry THF (25 mL) at 0° C. 3-Bromoprop-1-yne (1.55 mL, 80%in toluene) was added slowly dropwise after 30 min at 0° C. under argongas. The reaction was stirred under argon and allowed to reach roomtemperature. After 3 h, the reaction was quenched with water andextracted with ethyl acetate (30 mL×3). The organic layers were combinedand washed twice with water (50 mL) and once with brine (50 mL). Theorganic layer was dried over Na₂SO₄ and concentrated to give the crudeproduct. Flash chromatography (Hex:EA/8:1) yielded 24 (1.06 g, 89%) asan oil. ¹H NMR (400 MHz, chloroform-d) δ 9.92 (s, 1H), 7.87-7.78 (m,2H), 7.55-7.47 (m, 2H), 4.39 (dd, J=2.5, 0.9 Hz, 2H), 2.28 (t, J=2.4 Hz,1H), 1.46 (s, 9H). ¹³C NMR (100 MHz, chloroform-d) δ 191.2, 153.1,147.6, 133.4, 130.2, 125.4, 82.1, 79.4, 72.4, 39.4, 28.2.

2-Azidoethyl 4-Methylbenzenesulfonate (25)

To an oven-dried 100 mL round-bottom flask purged with argon gas andfitted with a magnetic stir bar was added DCM (15 mL), 2-azidoethan-1-ol(2.3 mL, 0.5 M in methyl tert-butyl ether), 4-toluenesulfonyl chloride(0.33 g), and Et₃N (0.32 mL). The reaction mixture was allowed to stirovernight at room temperature, and water was then added followed by twoextractions with DCM (30 mL). The organic layers were combined andwashed with water (50 mL) and brine (50 mL). Flash chromatography(Hex:EA/4:1) yielded 25 (0.22 g, 79%). ¹H NMR (400 MHz, chloroform-d) δ7.85-7.76 (m, 2H), 7.42-7.34 (m, 2H), 4.15 (ddd, J=5.2, 4.5, 0.9 Hz,2H), 3.53-3.42 (m, 2H), 2.45 (s, 3H).

Animal Preparation and Studies

All animal experiments were performed in accordance with guidanceprotocol approved by the Institutional Animal Care and Use Committee(IACUC) of Case Western Reserve University (protocols 2016-0028,2016-0023). The 8-week old WT C57BL/6 mice were used for all of the invitro and ex vivo tissue staining and SD rats (Harlan Laboratory,Indianapolis, Ind.) and Shiverer mice (Jackson Laboratory, Bar Harbor,Me.) were used for microPET/CT imaging studies. The animals were fastedovernight prior to imaging but had access to water. Their diet was thenreplenished after microPET/CT imaging.

Brain Focal Demyelination Rat Model

Sprague-Dawley female rats (220-250 g, 8 weeks old) were anesthetizedand positioned in a stereotaxic frame (Stoelting). A small incision wasmade in the scalp, and the corpus callosum was targeted using thefollowing stereotaxic coordinates, relative to bregma:anterior-posterior, 0.0 mm; medial-lateral, 2.0 mm; and dorsal-ventral,3.4 mm A small hole was drilled in the skull, and a 26S-gauge needleattached to a 10 μL Hamilton Syringe was lowered into the corpuscallosum according to the dorsal-ventral coordinate. A microinjectorpump (Stoelting) controlled the infusion of 6 μL of lysolecithin (LPC,0.1% in saline) at a rate of 0.25 μL/min, after which the needle wasleft in place for 2 min in order to prevent liquid reflux out of thebrain parenchyma. The incision was then closed using 5-0 Ethiconsutures, and the animals were allowed to recover on a heating pad. After5-7 days, the animals were ready for study.

Rat SCI Model

Sprague-Dawley female rats (220-250 g, 8 weeks old) were anesthetizedand a restricted laminectomy was conducted to expose the dorsal surfaceof T13. The vertebral column between T12 and L1 was then stabilized withclamps and forceps fixed to the base of an Infinite Horizon impactdevice. The midpoint of T13 was impacted with a force of 250 kDyn usinga 2.5 mm stainless steel impactor tip, which was used to induce amoderately severe contusive injury to the spinal cord. The musculaturewas then sutured over the laminectomy site and the skin closed withwound clips followed by subsequent treatment with Marcaine at theincision site. The force/displacement graph was used to monitor impactconsistency. After surgery, the animals were carefully monitored dailyfor pain and body weight with manual bladder expression 2-3 times dailyto stimulate reflex voiding until the animals could urinateindependently.

In Vitro Tissue Staining of Brain

Wild-type mice (20-22 g, 8 weeks old) were deeply anesthetized andperfused with precooled saline (4° C., 10 mL/min for 1 min followed by 7mL/min for 6 min), which was followed by fixation with precooled 4% PFAin PBS (4° C., 10 mL/min for 1 min followed by 7 mL/min for 6 min).Brain tissues were then removed, postfixed by immersion in 4% PFAovernight, dehydrated in 10%, 20%, and 30% sucrose solution, embedded ina freezing compound (OCT, Fisher Scientific, Suwanee, Ga.), andsectioned at 20 μm increments with a cryostat (Thermo HM525, ThermoFisher Scientific Inc., Chicago, Ill., USA). To provide stainingsections for preliminary FIR measurement, brain sections were collectedfrom AP (1.0) to AP (−0.1), and the first 12 sections were mounted inorder on the bottom of 12 superfrost slides (Fisher Scientific) with onesection on each slide. Sections 13-24 were mounted in order on themiddle of each slide, and sections 25-36 were mounted in order on thetop of each slide. Sections were then incubated with tested compounds (1mM, 5% DMSO in 1×PBS (pH 7.0), 6 sections per compound) for 25 min atroom temperature in the dark. Excess compounds were washed by brie yrinsing the slides in PBS (lx) and coverslipped with fluoromount-Gmounting media (Vector Laboratories, Burlingame, Calif.). Sections werethen examined under a microscope (Leica DM4000B, Leica Microsystem Inc.,Buffalo Grove, Ill., USA) equipped for fluorescence (DFC7000T), andimages of the stained mouse whole brain sections were acquired with thesame exposure time.

FIR Measurement

ImageJ software was used to quantify fluorescent intensity on sixsections of each tested compound. A ROI was selected on the genus of thecorpus callosum (gcc, white matter), and the same size of ROI wasapplied on the midline between gcc and the edge of the section (see FIG.8A), which is considered to be gray matter. Images were analyzed by twoexperienced individuals. The FIR values of white matter to gray matterwere then calculated.

Ex Vivo Tissue Staining

Wild-type mice were administered the newly synthesized compounds (40mg/kg) via tail vein injection, and 30-60 min later the mice wereperfused transcardially with saline followed by 4% PFA in PBS. Brainsand spinal cords were then removed, postfixed by immersion in 4% PFAovernight, dehydrated in 30% sucrose solution, cryostat sectioned at 100μm, mounted on superfrost slides, and images were acquired directlyusing a Leica fluorescent microscope.

In Vitro Staining of Rat Brain Treated with LPC

Five days after surgery, the rats were anesthetized and perfused withsaline followed by 4% PFA. Brain tissues were then removed, postfixed in4% PFA overnight, dehydrated in 10%, 20%, and 30% sucrose solution,embedded in OCT, and sectioned at 20 μm with a cryostat. To determine ifthe selected compound can differentiate demyelinated regions from normalmyelinated sheaths, we conducted in vitro tissue staining using spinalcord sections taken from LPC-treated rats. LPC-treated spinal cordsections were then incubated with tested compounds (1 mM, 5% DMSO in1×PBS (pH 7.0)) for 25 min at room temperature in the dark. Excesscompounds were washed by brie y rinsing the slides in PBS (lx) andcoverslipped with fluoromount-G mounting media (Vector Laboratories,Burlingame, Calif.). Sections were then examined under a microscope(Leica DM4000B, Leica Microsystem Inc., Buffalo Grove, Ill., USA)equipped for fluorescence (DFC7000T), and images of the stained mousewhole brain sections were acquired with the same exposure time. In themeantime, standard Luxol-Fast-Blue (LFB) staining was performed on theadjacent LPC-treated spinal cord section for comparison.

Radiosynthesis

No carrier-added (nca) [¹⁸F] fluoride was produced by a cyclotron(Eclipse High Production, Siemens) via the nuclear reaction ¹⁸O(p,n)¹⁸F. At the end of bombardment (EOB), the activity of aqueous [¹⁸F]uoride (50-100 mCi) was transferred to the GE Tracerlab F×n synthesizerby high helium pressure. After delivery, the radioactive solution waspassed through a Sep-Pak light QMA cartridge (Waters, preconditionedwith 5 mL of water followed by 10 mL of air in a syringe) and was elutedby K₂CO₃ solution (6 mg in 0.6 mL of water) followed by K₂₂₂ solution(12 mg in 1 mL of acetonitrile). The solvent was evaporated under asteam of helium at 85° C. for 5 min, and the residue was vacuumed at 55°C. for another 3 min to get the anhydrous K₂₂₂/[¹⁸F] complex. A solutionof the tosylated precursors (25, 3-5 mg, in 0.5 mL of acetonitrile) wasadded to the above dried complex, and the mixture was heated at 95° C.for 10 min. Cooling water (10 mL) was then added to the reaction vessel,and the mixture was passed through a C18 plus cartridge and an Oasis HLBplus cartridge in series (Waters, preconditioned with acetonitrile (10mL), water (10 mL), and followed by air (˜10 mL)). An additional 20 mLof water was used to rinse the reaction vial and the cartridges,followed by air (˜20 mL) to remove the residual water from the HLBcartridge. DMF (400 μL+500 μL) was used to elute [¹⁸F]26 from the WatersHLB cartridge into a reaction vial prefilled with a mixture of the clickreaction precursor 9 (5 mg), 1.25 mg of CuSO₄.5H₂O, 4 mg of sodiumascorbate, and 3 mg of BPDS in 50 μL (water/DMF=4/1). The mixture wasthen stirred for 10 min at 90° C. After cooling, 0.5 mL of HCl (1 M) wasadded and the resulting mixture was heated at 90° C. for 10-20 min. ANaOH solution (0.5 mL, 1 M) and water (15 mL) were then added, and theresulting mixture was passed through a preconditioned Sep-Pak C-18cartridge. The cartridge was then washed with another 20 mL of water,and the crude products were eluted with 1 mL of acetonitrile which wasfurther purified by semipreparative HPLC (Phenomenex C-18, 10 mm×250 mm;acetonitrile:H₂O=65:35; flow rate of 3 mL/min). The radioactive fractioncontaining the desired products was collected, diluted with water,loaded onto a Sep-Pak C-18 cartridge, and eluted with 1 mL of ethanol.After evaporation, the residue was redissolved in 5% ethanol in salinesolution and filtered (0.22 μm) into a sterile injection bottle foranimal use. RCP and specific activity (SA) were determined by analyticalHPLC (Phenomenex C-18; 4.6 mm×250 mm; acetonitrile:H₂O=65:35; flow rateof 1 mL/min). SA was calculated from the area of the UV peak of purifiedF-18 compound and titrated with the standard curve of the nonradioactivereference compound of known concentration.

In Vivo MicroPET/CT Imaging Studies

MicroPET/CT imaging was performed using a Siemens Inveon microPET/CTscanner in the Case Center for Imaging Research. For better anatomiclocalization, CT co-registration was applied. Before microPET imaging,CT scout views were taken to ensure the brain tissues were placed in theco-scan field of view (FOV) where the highest image resolution andsensitivity are achieved. Under anesthesia, radiotracer was administeredvia tail vein injection and immediately followed by a dynamic PETacquisition up to 60 min. After the microPET acquisition was done, therat was moved into the CT field and a two-bed CT scan was performed. Atwo-dimensional ordered subset expectation maximization (OSEM) algorithmwas used for image reconstruction using CT as attenuation correction.For quantitative analysis, the resultant PET images were registered tothe CT images, which enabled us to accurately define the ROI andquantify the radioactivity concentrations. In this study, the wholebrains of rats were used as ROI and the radioactivity concentrationswere determined in terms of SUVs.

Coregistration of In Situ 3D Cryoimaging with PET/CT Imaging

After microPET imaging studies, the rats were administered compound 21(3-5 mg) via tail vein injection, and 60 min later the rats wereperfused transcardially with saline followed by 4% PFA in PBS. Spinalcords were then removed, postfixed by immersion in 4% PFA overnight,dehydrated in 30% sucrose solution, cryostat sectioned at 100 μm,mounted consecutively on superfrost slides, and images were sequentiallyacquired directly using a Leica fluorescent microscope to give a seriesof 2D microscopic images along the length of the spinal cord. Sliceswere aligned sequentially using a semiautomated image registrationalgorithm. Starting from the first slice image, the current slice wasused as the reference image for alignment and the next slice was thefloating image. The floating image was aligned via control point pairsbetween corresponding anatomic features in the two images. A 2Drigid-body transformation (translation, rotation) was used to transformthe floating image to align with the reference and a minimum of threecontrol point pairs were used for the transformation. Once two sliceswere aligned, the algorithm continued with the newly aligned imageserving as the reference image and the next slice in the sequenceserving as the floating image. All slices in the sequence were alignedby this technique, giving a 3D reconstructed fluorescence image volume.Image volumes from fluorescence, PET, and CT were visualized in Amirausing volume rendering and aligned based on the shape of the spine andfiducials from the vertebrae.

In Situ Autoradiography

Wild-type mice were euthanized at 30 min post iv injection of [¹⁸F]21(111 MBq, 3.0 mCi). The brains were rapidly removed, placed in OCTembedding medium, and frozen at −20° C. After reaching equilibrium atthis temperature, the brains were coronally sectioned at 60 μm on acryostat and mounted on superfrost slides. After drying by air at roomtemperature, the slides were put in a cassette and exposed for 20 min tofilm to obtain images.

Results Chemical Synthesis

A series of fluorinated triazole derivatives of MeDAS with differentN-alkyl groups were of designed and synthesized as shown in Scheme 1.Incorporation triazole provides a pharmacophore that can be readilysynthesized and radiolabeled through a click reaction. Currently, themost common method of F-18 labeling is the direct nucleophilicsubstitution of sulfonic acid esters (such as tosylates, triflates, ormesylates) into the precursor with fluorine-18, but this reactioncondition raises the possibility of side reactions like E-2 eliminationand substitution of other potential leaving groups. Over the pastdecade, a 1,3-dipolar cycloaddition reaction between alkynes and azideshas been widely applied to radiolabeling with high specificity and goodyields, which thereby allows efficient F-18 labeling ofradiopharmaceuticals.

As shown in Scheme 1, the synthesis starts with Boc protection of theamino group of compound diethyl (4-aminobenzyl) phosphonate (1) togenerate compound 2, followed by alkylation with iodoalkanes to obtainvarious alkylated tert-butyl(4-((diethoxyphosphoryl)methyl)-phenyl)-carbamate (3-5). The obtainedcompounds 3-5 were coupled with corresponding aldehydes through theHorner-Wads-worth-Emmons reaction to generate N-alkylated tert-butyl(E)-(4-(4-(prop-2-yn-1-yloxy)styryl)phenyl)carbamate (6-8) having apropargyloxyl group in 72-74% yield, and N-alkylated tert-butyl(E)-(4-(4-((tert-butoxycarbonyl)(prop-2-yn-1-yl)-amino)styryl)phenyl)carbamate(9-11) with a Boc-protected propargylamino group in 68-72% yield. Clickreactions between compounds 6-11 and 1-azido-2-fluoroethane catalyzed bycopper(I) yielded fluorinated triazoles (12-17) in 50-60% yield, whichunderwent Boc-deprotection to give compounds 18-23 as the nal productsin 70-75% yield.

Fluorescence Properties

The excitation and emission spectra (0.3 mM in dichloromethane (DCM)) ofthe newly synthesized compounds were recorded using a Cary Eclipsefluorescent spectrophotometer. As shown in FIG. 7, all compounds werefluorescent with excitation wavelengths in the range of 300-415 nm andemission wavelengths in the range of 394-600 nm.

In Vitro Staining of Intact Myelin

The newly synthesized compounds were first examined for myelin bindingby in vitro staining of frozen mouse brain tissue sections. Because ofthe inherent fluorescence, the myelin-binding properties were directlyexamined by fluorescent microscopy after staining with each testedcompound at 10 μM concentration. All compounds labeled intact myelinsheaths present in the white matter included the corpus callosum,striatum, and cerebellum (FIG. 8A,B). Because myelin is rich in whitematter and deficient in gray matter, the difference of staining betweenthe white matter and gray matter is expected to reflect bindingspecificity of the test compounds. We thus calculated the fluorescentintensity ratio (FIR) of each test compound in the same region ofinterest (ROI) between the white matter and gray matter using ImageJ. Asshown in FIG. 8G, ROIs of same size were drawn on a representativeregion in the genus of the corpus callosum (gcc) and in the subcorticalgray matter (cortex), respectively. The newly synthesized compounds fellinto two categories based on the calculated FIR (FIG. 8F). The threeO-linked phenyl ring compounds(E)-4-(4-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)styryl)-N-alkane-aniline(18-20) showed FIRs ˜1. The other three N-linked phenyl ring compounds(E)-N-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-(4-(alkane-amino)styryl)-aniline(21-23) showed FIRs ˜1.5. The relatively higher FIRs indicated a higherdegree of specific binding. This study suggests that an N-linked phenylring is more myelin-specific than an O-linked phenyl ring in terms ofbinding to myelin sheaths.

Lipophilicity

To penetrate the blood-brain barrier (BBB), lipophilicity is one of thethree primary requirements that small molecules have to meet. Researchshowed that a lipophilicity value of 1.0-3.5 is ideal for a smallmolecule to cross the BBB. On the basis of the structures, we calculatedthe lipophilicity of the newly designed compounds as shown in Table 1.The C log P value of N-linked compound 21 is 2.86, which is very similarto our model compound MeDAS (2.91). Thus, compound 21 was selected forfurther evaluation.

TABLE 2 Compound Structure Log P 18

3.66 19

4.12 20

4.65 21

2.86 22

3.38 23

3.91

In Vitro Detection of Focal Demyelination in Rats

We then examined if compound 21 can be used to detect demyelinatedlesions present in a rat model of focal demyelination. In this model,demyelination was induced by lysolecithin (LPC), which wasstereotaxically injected into the corpus callosum of the righthemisphere. As shown in FIG. 8C, compound 21 was capable of detectingthe demyelinated focal lesion by fluorescent staining. The same foci ofdemyelination in the adjacent section were confirmed by conventionalLuxol-Fast-Blue (LFB) and cresyl violet staining (FIG. 8D). This resultdemonstrates that compound 21 can be used to detect brain lesions basedon specific binding to myelin.

In Situ Staining of Compound 21

Following the in vitro studies, we then investigated the ability ofcompound 21 to readily enter the brain and bind to myelin in situ. Adose of 1.0 mg of compound 21 (50 mg/kg) was administered via the tailvein to wild-type (WT) mice. One hour after injection, the mice wereperfused with saline followed by 4% paraformaldehyde (PFA) and the brainand spinal cord were removed and sectioned. The distribution of compound21 was then directly examined under a fluorescent microscope. As shownin FIG. 8E,F, strong fluorescence was visualized in the myelinatedregions including the corpus callosum, striatum, cerebellar whitematter, and the white matter of spinal cord. These studies suggest thatcompound 21 readily entered the brain and spinal cord and localized inall the myelinated regions in proportion to the myelin content.

Radiosynthesis

Encouraged by the in vitro and in situ results, we next evaluated the invivo pharmacokinetic profiles of compound 21 after labeling withpositron-emitting nuclide fluorine-18. As shown in Scheme 2, theradiosynthesis of [¹⁸F]21 was accomplished through a click reactionbetween compound 9 and 2-[¹⁸F] fluoroethylazide ([¹⁸F]26). [¹⁸F]26 wasprepared through a nucleophilic substitution of tosylate compound 25with [¹⁸F]KF in the presence of K₂CO₃ and Kryptofix (K₂₂₂) in MeCN at95° C. for 10 min. The identity of the product was confirmed usinganalytical HPLC by coinjection with cold standard compound 21. Theradio-chemical purity (RCP) of the final products was >98%, determinedby analytical radio-HPLC. The specific activity at the end of synthesiswas in the range of 1-2.5 Ci/μmol.

Quantitative MicroPET/CT Imaging Studies in WT Rats

Following radiosynthesis, the pharmacokinetic profile of [¹⁸F]21 wasfully characterized by quantitative microPET/CT imaging in WT rats(n=3). As shown in FIG. 9, [¹⁸F]21 exhibited high uptake in the brain.The brain radioactivity peaked at 3 min postinjection followed by rapidclearance before reaching a plateau at 40-60 min postinjection. Thetime-radioactivity curve fitted well the equation C(SUV) 1.1189e−0.0514t+0.3041, with a calculated time constant of clearance of 19.45min.

In Situ Autoradiography

To validate the PET results and confirm that the PET signals were indeedfrom specific binding to myelin, we conducted ex vivo autoradiography inthe mouse brain following administration of [¹⁸F]21 through tail veininjections. After 1 h, the mouse brain was dissected for coronal tissuesectioning. As shown in FIG. 9E, [¹⁸F]21 distinctly labeled the corpuscallosum, which is the region with a high density of myelin sheaths. Theautoradiographic visualization showed that the distribution of [¹⁸F]21was consistent with the histological staining of myelinated regions withthe corresponding nonlabeled compound 21 (see FIG. 8A). Thus,combination of in situ autoradiography and PET imaging confirmed that[¹⁸F]21 binds specifically to myelin in vivo.

Quantitative MicroPET/CT Imaging Studies in Shiverer Mice

To further evaluate the in vivo binding specificity of [¹⁸F]21, weinvestigated the pharmacokinetic profiles of [¹⁸F]21 in a Shiverer mousemodel that is deficient in myelin in the brain. Thus, thepharmacokinetic profiles of [¹⁸F]21 were quantitatively compared betweenShiverer mice (C3Fe.SWV-Mbpshi/J, n=2) and age-matched WT mice (CB57J,n=2).

Dynamic emission scans were acquired for 60 min in 3D list modeimmediately after [¹⁸F]21 (0.3 mCi each mouse, 11.1 MBq) wasadministered. The brain uptake of [¹⁸F]21 was lower in the Shivererbrain than in the WT littermates (1.25 vs 1.64). After the brainconcentration of [¹⁸F]21 reached a plateau, the SUVs from 40 to 60 minwere summarized and compared. As shown in FIG. 10, [¹⁸F]21 uptake in theShiverer mouse brain was significantly decreased compared to the controlmouse brain. These results suggested that [¹⁸F]21 indeed binds to myelinin the brain with high specificity.

Quantitative MicroPET/CT Imaging Studies in the Spinal Cord

So far, imaging of the spinal cord remains a great challenge. Ourstudies showed that compound 21 is capable of detecting focaldemyelinated lesions induced in the rat brain in vitro. Next, we askedwhether [18F]21 PET is capable of in vivo imaging of myelin deficiencyor myelin damage in living animal models. We conducted [¹⁸F]21-PETimaging in a rat model of thoracic contusive SCI. In this model, thecontusion was made at T13 to introduce demyelination. In vivo [¹⁸F]21PET imaging was performed before (as a baseline scan) and 1 day aftercontusion. The uptake of [¹⁸F]21 in every spine segment was thennormalized to the average spine uptake. As shown in FIGS. 11A, B, thewhole intact thoracic region of the spinal cord could be clearlyvisualized by [¹⁸F]21-PET imaging in the control rats, but the lesion atvertebrae level T13 was clearly visualized by [¹⁸F]21-PET in the SCImodel (FIGS. 11C,D). Quantitative analysis of [¹⁸F]21 uptake in thewhole thoracic region (T1-T13), and part of the lumbar vertebra showeddistinct patterns of uptake. As shown in FIG. 11E, the uptake of [¹⁸F]21in T13 in the SCI group was 0.81, which was significantly lower thanthat in their own baseline scans (1.02). After microPET/CT imaging, weconducted in situ histological staining of the spinal cord 60 min afterinjection of nonlabeled compound 21 through the tail vein. As shown inFIG. 12, in situ histological staining of the SCI (T13) tissue sectionwith compound 21 showed that a demyelinated lesion was present at thedorsal column (A), which was confirmed by LFB and cresyl violet stainingof the adjacent sections (B).

Coregistration of In Situ 3D Cryoimaging with MicroPET/CT Imaging

To confirm the in vivo imaging of the spinal white matter by PET/CT, weacquired 2D fluorescence images and generated a 3D reconstructed imagestack. Thus, nonlabeled compound 21 was administered through tail veininjection into the rat after microPET/CT imaging of the spinal cord ofSCI rats. One hour later, the rat was perfused with saline followed by4% paraformaldehyde (PFA) and the spinal cord (T7-L2) was removed,sequentially sectioned, and imaged using a Leica fluorescent microscope.

Slices were aligned sequentially using a semiautomated imageregistration algorithm through Matlab, giving a 3D reconstructedfluorescence image volume. Image volumes from fluorescence, PET, and CTwere visualized in Amira using volume rendering and aligned based on theshape of the spine and fiducials from the vertebrae. As shown in FIG.13, 3D reconstructed fluorescence images were coregistered withmicroPET/CT images, where the reduced fluorescence signal was consistentwith the reduced microPET signal in the same spinal cord region T13,thereby confirming that the reduced PET signal at the injury site wasactually caused by demyelination after the T13 contusion.

DISCUSSION

We developed a novel F-18-labeled radiotracer [¹⁸F]21 that can be usedto quantitatively monitor myelin content in vivo. F-18-labeledradiotracers are often used to facilitate remote distribution of thetracer from a centralized radiopharmacy production site. On the basis ofour previously developed C-11 myelin-imaging agent, MeDAS, we designedand synthesized a series of fluorinated analogues for SAR studies inorder to identify lead candidates capable of in vivo imaging of myelin.In this work, we designed and synthesized a series of compounds byalkylating one of the terminal amino groups. Our previous studiessuggested that the two terminal amino groups of MeDAS are essentialmoieties for specific binding to myelin. Alkylation of the amino groupsdoes not adversely alter the binding properties, which makes it possibleto introduce fluorine to the pharmacophore.

We first examined the lipophilicity of the newly synthesized compounds,which has a significant impact on brain permeability. Becauseintroduction of fluorine to one amino group often renders the compoundless lipophilic, we designed a series of fluorinated compounds byintroducing an alkyl group into the other amino group to compensate forthe decreased lipophilicity. The fluoro group was introduced through aCu(I)-catalyzed Huisgen cycloaddition, known as the “click reaction”,which can be conducted under mild conditions with high yield.

After synthesis of compounds 18-23, we conducted in vitro staining ofmouse brain tissue sections. The binding specificity for myelin of eachcompound was estimated by calculating the FIR between the myelin-richwhite matter and myelin-deficient gray matter. As shown in FIG. 7, thesecompounds had similar fluorescence properties (same excitation andemission wavelength), making it possible for direct comparison of myelinbinding specificity based on the FIR when tissue staining was conductedusing the same thickness of sections and the same exposure time of imageacquisition. Our study suggested that N-linked analogues (21-23) exhibithigher myelin binding specificity than O-linked analogues (18-20). Thismay be in part due to the fact that introduction of 0 to the moleculesrenders the compounds more lipophilic as is evidenced by the higher LogP values shown in Table 1, which increase nonspecific binding.

Among those compounds with higher FIR, compound 21 was selected forfurther study as it has a log Poct value in the range between 1.5 and3.5, which is often required for optimal brain uptake. Combination ofthese studies led us to focus on compound 21 for further in situstaining, which is usually a binding specificity of fluorescentcompounds. Our studies indicate that compound 21 readily enters thebrain and specifically binds to myelin in white matter regions such asthe corpus callosum, striatum, and spinal cord. Furthermore, compound 21is capable of detecting demyelinated lesions in a rat model of focaldemyelination induced by LPC. These studies further confirmed that thecharacteristic brain uptake and staining of compound 21 is proportionalto myelin content in the CNS.

Following in vitro and ex vivo studies, we proceeded with radiosynthesisof [¹⁸F]21 for in vivo PET imaging studies. The radiosynthesis wasachieved by coupling 2-[¹⁸F] fluoroethyl azide with its alkyne precursorcompound 9 in the presence of excess Cu²⁺/ascorbate andbathophenanthrolinedisulfonic acid disodium salt (BPDS) in aqueoussolution, yielding the 1,2,3-triazoles product with the desiredradiochemical yield (80-90%, n=5, decay corrected) and radiochemicalpurity of over 98%. 2-[¹⁸F] fluoroethyl azide is the key intermediate.Although the entire radiosynthesis could be conducted in a “one-pot”fashion, the radiochemical yield was relatively low. Separate isolationand purification of 2-[¹⁸F] uoroethyl azide was found to be critical forsuccessful radiolabeling. Although [¹⁸F]-fluoroethyl azide is highlyvolatile with a boiling point of 50° C., distillation of [¹⁸F]fluoroethyl azide from the reaction mixture was not practical. We thusused solid phase extraction (SPE) instead with two series of WatersOasis HLB cartridges to successfully trap [¹⁸F] uoroethyl azide withhigh yield and purity.

Following radiosynthesis, we conducted quantitative micro-PET/CT imagingstudies in wild-type rats. An ideal radiotracer for in vivo PET imagingof myelin should meet several criteria such as high brain uptake andprolonged retention due to high myelin-binding potential. As expected,quantitative PET data analysis showed a high and rapid accumulation of[¹⁸F]21 in the brain followed by a rapid nonspecific binding clearance.In situ autoradiography further confirmed that the radioactivity signaldetected by PET indeed reflects the specific spatial distribution of[¹⁸F]21 after binding to myelinated fibers. The combination of in situautoradiography with [¹⁸F]21 and with PET imaging studies confirmed that[¹⁸F]21 is a specific imaging marker of myelin content.

Given the fact that [¹⁸F]21 binds to myelin sheaths in vivo, we furtherdetermined whether [¹⁸F]21-PET is capable of imaging myelin deficiencyor damage as tested in two animal models. The Shiverer mouse model wasused to represent a myelin-deficiency in the brain, while the rat modelwith traumatic SCI was used to represent myelin damage in the spinalcord. As shown in FIG. 11, quantitative analysis of the uptake of[¹⁸F]21 in the brain showed that retention in the Shiverer mice wassignificantly lower than that in the WT control (p=0.00028, two-tailed ttest). MicroPET/CT imaging in the SCI rat model further confirmed that[¹⁸F]21 is capable of detecting and quantifying myelin damage in thespinal cord. As shown in FIG. 12, a distinct myelin-imaging defect atT13 contused tissue could be visualized and quantified directly byPET/CT imaging. Before contusion, [¹⁸F]21 uptakes at T12, T13, and L1spine segments were respectively 0.99, 1.02, and 1.05, while the uptakeswere 0.99, 0.81, and 1.08 l day post-T13 contusion. [¹⁸F]21 uptakedecreased dramatically at the site of injury (T13) compared with its ownbaseline scan.

To date, to expedite the translation of myelin imaging into clinicalpractice, some radiotracers such as PIB and florbetaben that wereoriginally developed for amyloid imaging have been repurposed for myelinimaging. These radiotracers have already been approved by the FDA forclinical use. Such radiotracers were found to bind to the myelin-richwhite matter in the brain, albeit to a lesser extent than amyloiddeposits. It is thus believed that they could directly be used in MSpatients to image myelin distribution in the white matter region whereno amyloid deposits would be present, even in Alzheimer's patients.Thus, the utility of these amyloid-imaging radiotracers were tested invarious subtypes of MS patients for correlation of tracer uptake withMRI characterization of brain lesions, disease progression patterns, andfunctional disability. These studies provided proof-of-the-concept thatPET imaging is a valid clinical imaging tool to observe white matterchanges in terms of myelin distribution. However, these radiotracersoriginally were so optimized for amyloid imaging that any bindingpotential in the white matter was minimized. This makes it difficult toaddress several important issues concerning imaging sensitivity andspecificity under an in inflammatory condition that is typical of MS.Independent studies often led to controversial interpretations ofimaging results.

To overcome these limitations and enhance the myelin-imaging sensitivityand specificity, new myelin-imaging radio-tracers must be developedwhereby the binding potential for myelin can be maximized in the whitematter. The parent compound, MeDAS, was thus optimized to be specificfor myelin and to minimize any effect of in inflammation. On the basisof solid preclinical validation, it will significantly improve theimaging sensitivity and specificity.

An important innovative aspect of this work lies in the combination ofPET/CT with 3D cryoimaging. PET is a tomographic, functional imagingmodality with high quantitative capacity. Yet, PET quantitative analysismust be validated by correlation with histological characterization.This is often a very challenging task, particularly in spinal cordimaging due to its small and mobile structure. Tissue preparation andsectioning are very time-consuming, and only selected tissue sectionscan be used for validation of PET-imaging results, which is likelybiased by the limited scope of sampling. Tomographic cryoimaging thuscan overcome this limitation as it can provide high-resolution,three-dimensional images of the entire spinal cord for histologicalcharacterization. The whole process can be fully automated using aCryoViz system (BiolnVision, Inc.). The imaging results are thus lesssubjective as the sampling bias can be essentially eliminated. Moreimportantly, the 3D cryoimaging of the spinal cord was carried out insitu using nonlabeled, otherwise identical compound 21, which wasadministered via tail vein injection in a way similar to the PET scan.This promotes accurate image coregistration between PET and cryoimagingfor quantitative analysis.

This new multimodality PET/cryoimaging technique was first applied inthe studies of SCI. In vivo imaging of myelin changes after SCI is ofparticular importance as demyelination and remyelination have directeffects on disability and functional recovery. To confirm that thecontusive injury at T13 was associated directly with demyelinationrather than tissue loss, we conducted in situ 3D cryoimaging withfluorescent compound 21 following microPET/CT imaging using [¹⁸F]21. Asshown in FIG. 12A, a distinct demyelination at the dorsal portion wasobserved. This was further confirmed by standard LFB and cresyl violetstaining of myelin sheaths on the adjacent sections, which suggeststhere was a significant demyelinated lesion in the white matter regionat the injury site. In contrast, no tissue loss was observed andmyelinated white matter outside the injured region remained intact (FIG.13B). After tomographic 3D reconstruction, the cryoimages werecoregistered with PET/CT images (FIG. 13). We observed significantreduction of the fluorescence signal in the lesion region of the spinalcord, validating the sensitivity and specificity of PET. These findingssuggest that [¹⁸F]21-PET imaging can be used as a diagnostic marker ofmyelin damage. In addition, we demonstrated, for the first time, thefeasibility of simultaneous coregistration of PET/CT with and validationby high-resolution, 3D cryoimaging, which allows for seamlesscombination of physiological information with the high sensitivity andquantification capability provided by PET and microscopic histologyprovided by cryoimaging. This newly developed multimodality imagingtechnique should greatly facilitate radiotracer development and efficacyevaluation of novel therapies.

In summary, a series of fluorinated fluorescent myelin-binding agentswas synthesized for PET and 3D cryoimaging. Following systematic SARstudies, we identified a novel myelin-imaging agent (TAFDAS, 21) thatreadily penetrates the BBB and binds to myelin membranes in the brainand spinal cord. Sequential [¹⁸F]21-PET imaging and 3D cryoimaging incontusion rat model of SCI demonstrate, for the first time, thatcombination of PET and cryoimaging is a new imaging tool with highsensitivity, specificity, and spatial resolution.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

1. A method of detecting myelin in vivo in a subject's tissue, themethod comprising: (i) administering to the subject a radioligandincluding a compound having the formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup; R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group; R³ and R⁴ are same or different and areeach independently H, NHR″, where R″ is H or a lower alkyl group, or aradiolabeled lower alkyl group, alkylene group, alkenyl group, alkynylgroup, or alkoxy group; R⁵ and R⁶ are H or are linked to form a cyclicring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, orheteroalicyclic; R⁷ and R⁸ are H or are linked to form a cyclic ring,wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, orheteroalicyclic; wherein at least one of R¹, R², R³, or R⁴ includes aradiolabel selected from the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and^(99m)Tc; or a pharmaceutically acceptable salt thereof; (ii) detectingthe location, distribution, and/or amount of the radioligand that isbound to and/or labels myelin to detect myelin in the tissue.
 2. Themethod of claim 1, wherein the radioligand has the formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup; R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group; R³ is H, or a radiolabeled lower alkylgroup, alkylene group, alkenyl group, alkynyl group, or alkoxy group;wherein at least one of R¹, R², or R³ includes a radiolabel selectedfrom the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or apharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein the radioligand has the formula:

wherein R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group; R³ is H, or a radiolabeled lower alkylgroup, alkylene group, alkenyl group, alkynyl group, or alkoxy group;wherein at least one of R² or R³ includes a radiolabel selected from thegroup consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or a pharmaceuticallyacceptable salt thereof.
 4. The method of claim 1, wherein theradioligand has the formula:

wherein R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group; X¹ is a lower alkyl group, alkylene group,alkenyl group, alkynyl group, or alkoxy group; Y¹ is a radiolabelselected from the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; ora pharmaceutically acceptable salt thereof.
 5. The method of claim 1,wherein the radioligand has the formula:

R² is H, or NHR′, where R′ is H, a lower alkyl group, or a radiolabeledtriazole group; n¹ is 1 to 6; Y¹ is a radiolabel selected from the groupconsisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or a pharmaceuticallyacceptable salt thereof.
 6. The method of claim 1, wherein theradioligand has the formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup; R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group; wherein at least one of R¹ or R² includes aradiolabel selected from the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and^(99m)Tc; or a pharmaceutically acceptable salt thereof.
 7. The methodof claim 1, wherein the radioligand has the formula:

wherein R² is H, or NHR′, where R′ is H or a lower alkyl group; n² is 1to 6; n³ is 1 to 6; Y¹ is a radiolabel selected from the groupconsisting of ¹⁸F, ¹²³I, ¹²⁵I, and ^(99m)Tc; or a pharmaceuticallyacceptable salt thereof.
 8. The method of claim 1, wherein theradioligand has the formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup; R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group; R³ and R⁴ are same or different and areeach independently H, NHR″, where R″ is H or a lower alkyl group, or aradiolabeled lower alkyl group, alkylene group, alkenyl group, alkynylgroup, or alkoxy group; wherein at least one of R¹, R², R³, or R⁴includes a radiolabel selected from the group consisting of ¹⁸F, ¹²³I,¹²⁵I, and ^(99m)Tc; or a pharmaceutically acceptable salt thereof. 9.The method of claim 1, wherein the radioligand has the formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup; R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group; wherein at least one of R¹ or R² includes aradiolabel selected from the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and^(99m)Tc; or a pharmaceutically acceptable salt thereof.
 10. The methodof claim 1, wherein the radioligand has the formula:

wherein R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group; R³ and R⁴ are same or different and areeach independently H, NHR″, where R″ is H or a lower alkyl group, or aradiolabeled lower alkyl group, alkylene group, alkenyl group, alkynylgroup, or alkoxy group; wherein at least one of R², R³, or R⁴ includes aradiolabel selected from the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and^(99m)Tc; or a pharmaceutically acceptable salt thereof.
 11. The methodof claim 1, wherein the radioligand has the formula:

or pharmaceutically acceptable salts thereof.
 12. The method of claim 1,the radioligand is detected by in Positron Emission Tomography (PET)imaging or micro Positron Emission Tomography (microPET) imaging. 13.The method of claim 1, further comprising the step of administering theradioligand to the subject parenterally.
 14. The method of claim 1,wherein the radioligand further comprises a chelating group or a nearinfrared imaging group.
 15. A method of detecting a myelin relateddisorder in a subject in need thereof, the method comprising the stepsof: (i) administering to the subject a radioligand that includes acompound having the formula:

wherein R¹ is a H, a lower alkyl group, or a radiolabeled triazolegroup; R² is H, or NHR′, where R′ is H, a lower alkyl group, or aradiolabeled triazole group; R³ and R⁴ are same or different and areeach independently H, NHR″, where R″ is H or a lower alkyl group, or aradiolabeled lower alkyl group, alkylene group, alkenyl group, alkynylgroup, or alkoxy group; R⁵ and R⁶ are H or are linked to form a cyclicring, wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, orheteroalicyclic; R⁷ and R⁸ are H or are linked to form a cyclic ring,wherein the cyclic ring is aromatic, alicyclic, heteroaromatic, orheteroalicyclic; wherein at least one of R¹, R², R³, or R⁴ includes aradiolabel selected from the group consisting of ¹⁸F, ¹²³I, ¹²⁵I, and^(99m)Tc; or a pharmaceutically acceptable salt thereof; (ii) detectingthe distribution and/or amount of the radioligand in a region ofinterest in tissue of the subject; and (iii) comparing the detecteddistribution and/or amount of the radioligand to a control, wherein adecreased distribution and/or amount of the radioligand compared to thecontrol is indicative of a decrease in myelination of the tissue and thepresence of myelination related disorder. 16-24. (canceled)
 25. Themethod of claim 15, wherein the radioligand has the formula:

or pharmaceutically acceptable salts thereof.
 26. The method of claim15, wherein the radioligand is detected using Positron EmissionTomography (PET) or micro Positron Emission Tomography (microPET)imaging modality.
 27. The method of claim 15, further comprising thestep of administering the radioligand to the subject parenterally. 28.The method of claim 15, wherein the radioligand further comprises achelating group or a near infrared imaging group.
 29. The method ofclaim 15, wherein the myelin related disorder is a neurodegenerativeautoimmune disease.
 30. The method of claim 29, wherein theneurodegenerative autoimmune disease is multiple sclerosis. 31-45.(canceled)